Compositions and Methods for Treating Proliferation Disorders

ABSTRACT

The present invention relates to methods of treating cell proliferative disorders, such as cancer or Proteus syndrome, by utilizing 3-(3-(4-(1-aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine or 3-(3-(4-(1-aminocyclobutyl)phenyl)-5-(3-morpholinophenyl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine or N-(1-(3-(3-(4-(1-aminocyclobutyl)phenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-5-yl)phenyl)piperidin-4-yl)-N-methylacetamide. The methods of the present invention can also relate to methods of treating cell proliferative disorders, such as cancer or Proteus syndrome, by utilizing the above compounds in combination with ((R)-6-(2-fluorophenyl)-N-(3-(2-((2-methoxyethyl)amino)ethyl)phenyl)-5,6-dihydrobenzo[h]quinazolin-2-amine).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S. Ser. No.62/046,502, filed Sep. 5, 2014 and U.S. Ser. No. 62/082,236, filed Nov.20, 2014. The contents of each of these applications are incorporated intheir entireties.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States,exceeded only by heart disease. (Cancer Facts and Figures 2004, AmericanCancer Society, Inc.). Despite recent advances in cancer diagnosis andtreatment, surgery and radiotherapy may be curative if a cancer is foundearly, but current drug therapies for metastatic disease are mostlypalliative and seldom offer a long-term cure. Even with newchemotherapies entering the market, the need continues for new drugseffective in monotherapy or in combination with existing agents as firstline therapy, and as second and third line therapies in treatment ofresistant tumors.

Cancer cells are by definition heterogeneous. For example, within asingle tissue or cell type, multiple mutational “mechanisms” may lead tothe development of cancer. As such, heterogeneity frequently existsbetween cancer cells taken from tumors of the same tissue and same typethat have originated in different individuals. Frequently observedmutational “mechanisms” associated with some cancers may differ betweenone tissue type and another (e.g., frequently observed mutational“mechanisms” leading to colon cancer may differ from frequently observed“mechanisms” leading to leukemias). It is therefore often difficult topredict whether a particular cancer will respond to a particularchemotherapeutic agent (Cancer Medicine, 5^(th) edition, Bast et al., B.C. Decker Inc., Hamilton, Ontario).

Components of cellular signal transduction pathways that regulate thegrowth and differentiation of normal cells can, when dysregulated, leadto the development of cellular proliferative disorders and cancer.Mutations in cellular signaling proteins may cause such proteins tobecome expressed or activated at inappropriate levels or atinappropriate times during the cell cycle, which in turn may lead touncontrolled cellular growth or changes in cell-cell attachmentproperties. For example, dysregulation of receptor tyrosine kinases bymutation, gene rearrangement, gene amplification, and overexpression ofboth receptor and ligand has been implicated in the development andprogression of human cancers.

AKT protein family, which members are also called protein kinases B(PKB) plays an important role in mammalian cellular signaling. Inhumans, there are three genes in the AKT family: Akt1, Akt2, and Akt3.These genes code for enzymes that are members of theserine/threonine-specific protein kinase family. Akt1 is involved incellular survival pathways, by inhibiting apoptotic processes. Akt1 isalso able to induce protein synthesis pathways, and is therefore a keysignaling protein in the cellular pathways that lead to skeletal musclehypertrophy, and general tissue growth. Akt2 is an important signalingmolecule in the Insulin signaling pathway and is required to induceglucose transport. The role of Akt3 is less clear, though it appears tobe predominantly expressed in brain.

The AKT family regulates cellular survival and metabolism by binding andregulating many downstream effectors, e.g. Nuclear Factor-κB, Bcl-2family proteins and murine double minute 2 (MDM2). Akt1 is known to playa role in the cell cycle. Moreover, activated Akt1 may enableproliferation and survival of cells that have sustained a potentiallymutagenic impact and, therefore, may contribute to acquisition ofmutations in other genes. Akt1 has also been implicated in angiogenesisand tumor development. Studies have shown that deficiency of Akt1enhanced pathological angiogenesis and tumor growth associated withmatrix abnormalities in skin and blood vessels. Since it can blockapoptosis, and thereby promote cell survival, Akt1 is a major factor inmany types of cancer.

Accordingly, there is a need in the art for new compounds and methodsfor modulating various genes and signaling pathways; and methods fortreating proliferation disorders, including cancer. The presentinvention addresses these needs.

SUMMARY OF THE INVENTION

The present invention provides a method of treating a cell proliferativedisorder, said method comprising administering, to a subject in needthereof, a therapeutically effective amount of a composition comprisingat least one of Compound 1

Compound 2

or Compound 3

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof, wherein said cell proliferative disorder is treated.

The cell proliferative disorder can be the result of a mutation in atleast one of AKT, PIK3CA or PTEN. The cell proliferative disorder can becancer. The cancer can be lung cancer, small cell lung cancer, non-smallcell lung cancer, colon cancer, breast cancer, pancreatic cancer,prostate cancer, anal cancer, renal cancer, cervical cancer, brain,gastric/stomach cancer, head and neck cancer, thyroid cancer, bladdercancer, endometrial cancer, uterine cancer, intestinal cancer, hepaticcancer, leukemia, lymphoma, T-cell lymphoblastic leukemia, primaryeffusion lymphoma, chronic myelogenous leukemia, melanoma, Merkel cellcancer, ovarian cancer, alveolar soft part sarcoma (ASPS), clear cellsarcoma (CCS), Paget's disease, rhabdomysarcoma, angiosarcoma,cholangiocarcinoma or hepatocellular carcinoma. The cancer can beendometrial cancer, ovarian cancer, primary effusion lymphoma, T-celllymphoblastic leukemia, rhabdomysarcoma, Paget's disease, angiosarcoma,pancreatic endocrine tumor, anal squamous cell carcinoma, Merkel cellcancer, hormone receptor positive breast cancer or luminal breastcancer, head and neck squamous cell carcinoma, lung squamous cellcarcinoma, gastric/stomach cancer, or thyroid cancer.

The cell proliferative disorder can be a non-cancer condition, diseaseor disorder. The non-cancer condition, disease or disorder can bepituitary adenoma, leishmaniasis, skin-related hyperproliferativedisorders, psoriasis, eczema, hyperpigmentation disorders, eye-relatedhyperproliferative disorders, age-related macular degeneration, Herpessimplex virus, Proteus syndrome (Wiedemann syndrome), macrodactylysyndrome, Harlequin ichthyosis, CLOVES syndrome, atopic dermatitis,LEOPARD syndrome, systemic sclerosis, Spinocerebullar ataxia type 1,fibroadipose hyperplasia, hemihyperplasia-multiple lipomatosis syndrome,megalencephaly, rare hypoglycemia, Klippel-Trenaunay syndrome,harmatoma, Cowden syndrome or overgrowth-hyperglycemia. The cellproliferative disorder can be pituitary adenoma, Proteus syndrome,fibroadipose hyperplasia, CLOVES syndrome, macrodactyly syndrome,Harlequin ichthyosis, LEOPARD syndrome, Herpes simplex virus,leishmaniasis, psoriasis, atopic dermatitis, Spinocerebullar ataxia type1, or systemic sclerosis.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing viability of Proteus cells in the presence ofserum and various dosages of Compound 1 after 72 hours of treatment.

FIG. 2 is a graph showing viability of Proteus cells in the presence ofserum and various dosages of Compound 1 after 24 hours of serumstarvation and 72 hours of treatment.

FIG. 3 is a graph showing viability of PIK3CA cells in the presence ofserum and various dosages of Compound 1 after 72 hours of treatment.

FIG. 4 is a graph showing viability of PIK3CA cells in the presence ofserum and various dosages of Compound 1 after 24 hours of serumstarvation and 72 hours of treatment.

FIGS. 5A and 5B are a series of graphs showing viability of Proteussingle cell clones in the presence or absence of serum and variousdosages of Compound 1 (FIG. 5A) or everolimus (FIG. 5B) after 24 hoursof serum starvation and 72 hours of treatment.

FIG. 6 is a graph showing the phosphorylation status of AKT1 in Proteussingle cell clones in the presence or absence of serum and variousdosages of Compound 1 after 24 hours of serum starvation and 24 hours oftreatment.

FIGS. 7A and 7B are a series of graphs showing the phosphorylationstatus of S6 in Proteus single cell clones in the presence (FIG. 7B) orabsence of serum (FIG. 7A) and various dosages of Compound 1 after 24hours of serum starvation and 24 hours of treatment.

FIGS. 8A and 8B are a series of graphs showing the phosphorylationstatus of AKT1 in four different Proteus cell lines from a singlepatient with differing AKT1 p.E17K in the presence (FIG. 8A) or absenceof serum (FIG. 8B) and various dosages of Compound 1 after 24 hours ofserum starvation and 24 hours of treatment.

FIGS. 9A and 9B are a series of graphs showing the phosphorylationstatus of S6 in four different Proteus cell lines from a single patientwith differing AKT1 p.E17K in the presence (FIG. 9B) or absence of serum(FIG. 9A) and various dosages of Compound 1 after 24 hours of serumstarvation and 24 hours of treatment.

FIG. 10 is a graph showing the phosphorylation status of AKT1 in cellsobtained from a patient with PIK3CA p.H1047R mutation (PS109.3) orcontrol cells (PS95.2) in the presence or absence of serum and variousdosages of Compound 1 after 24 hours of serum starvation and 24 hours oftreatment.

FIGS. 11A and 11B are a series of graphs showing the phosphorylationstatus of S6 in cells obtained from a patient with PIK3CA p.H1047Rmutation (PS109.3) or control cells (PS95.2) in the presence (FIG. 11B)or absence of serum (FIG. 11A) and various dosages of Compound 1 after24 hours of serum starvation and 24 hours of treatment.

FIG. 12 is a graph showing the phosphorylation status of AKT1 in cellsobtained from a patient with PIK3CA p.H1047L mutation (PS129.3, GSA) orcontrol cells (PS75.1) in the presence or absence of serum and variousdosages of Compound 1 after 24 hours of serum starvation and 24 hours oftreatment.

FIGS. 13A and 13B are a series of graphs showing the phosphorylationstatus of AKT1 in cells obtained from a patient with PIK3CA p.H1047Lmutation (PS129.3, GSA) or control cells (PS75.1) in the presence (FIG.13B) or absence of serum (FIG. 13A) and various dosages of Compound 1after 24 hours of serum starvation and 24 hours of treatment.

FIGS. 14A, 14B, 14C, and 14D are a series of graphs showing thephosphorylation status of AKT1 in Proteus single cell clones in thepresence (FIGS. 14C and 14D) or absence of serum (FIGS. 14A and 14B) and125 nM of Compound 1 after 24 hours of serum starvation and at varioustreatment timepoints.

FIG. 15 is a graph showing the phosphorylation status of AKT1 in Proteussingle cell clones in the presence or absence of serum and variousdosages of everolimus after 24 hours of serum starvation and 24 hours oftreatment.

FIGS. 16A and 16B are a series of graphs showing the phosphorylationstatus of S6 in Proteus single cell clones in the presence (FIG. 16B) orabsence of serum (FIG. 16A) and various dosages of everolimus after 24hours of serum starvation and 24 hours of treatment.

FIG. 17 is a series of photographs the effect of Compound 1 on pAKT andpPRAS40 in KU-19-19 and AN3CA cells at various dosages following twohour treatment.

FIG. 18 is a series of photographs the effect of Compound 1, MK-2206 andGDC0068 on pAKT and pPRAS40 in KU-19-19 cells at various dosagesfollowing two hour treatment.

DETAILED DESCRIPTION OF THE INVENTION 1. Methods of Treatment

The present invention provides methods for the treatment of a cellproliferative disorder in a subject in need thereof by administering toa subject in need of such treatment, a therapeutically effective amountof a composition comprising at least one of Compound 1, Compound 2 orCompound 3, or a pharmaceutically acceptable salt, solvate, hydrate, orprodrug thereof, wherein said cell proliferative disorder is treated.The cell proliferative disorder can be cancer, a precancerous conditionor a non-cancer condition, disease or disorder. The present inventionfurther provides the use of a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, for the preparation of a medicament useful for thetreatment of a cell proliferative disorder.

The present invention also provides methods of protecting against a cellproliferative disorder in a subject in need thereof by administering toa subject in need of such treatment, a therapeutically effective amountof a composition comprising at least one of Compound 1, Compound 2 orCompound 3, or a pharmaceutically acceptable salt, solvate, hydrate, orprodrug thereof, wherein said cell proliferative disorder is treated.The cell proliferative disorder can be cancer, a precancerous conditionor a non-cancer condition, disease or disorder. The present inventionalso provides the use of compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, for the preparation of a medicament useful for theprevention of a cell proliferative disorder.

As used herein, a “subject in need thereof” is a subject having a cellproliferative disorder, or a subject having an increased risk ofdeveloping a cell proliferative disorder relative to the population atlarge. A subject in need thereof can have a precancerous condition.Preferably, a subject in need thereof has cancer. A “subject” includes amammal. The mammal can be e.g., any mammal, e.g., a human, primate,bird, mouse, rat, fowl, dog, cat, cow, horse, goat, rabbit, camel, sheepor a pig. Preferably, the mammal is a human.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe invention encompass a variety of conditions wherein cell division isderegulated. Exemplary cell proliferative disorder include, but are notlimited to, neoplasms, benign tumors, malignant tumors, pre-cancerousconditions, in situ tumors, encapsulated tumors, metastatic tumors,liquid tumors, solid tumors, immunological tumors, hematological tumors,cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidlydividing cells. The term “rapidly dividing cell” as used herein isdefined as any cell that divides at a rate that exceeds or is greaterthan what is expected or observed among neighboring or juxtaposed cellswithin the same tissue. A cell proliferative disorder includes aprecancer or a precancerous condition. A cell proliferative disorderincludes cancer. A cell proliferative disorder includes a non-cancercondition or disorder. Preferably, the methods provided herein are usedto treat or alleviate a symptom of cancer. The term “cancer” includessolid tumors, as well as, hematologic tumors and/or malignancies. A“precancer cell” or “precancerous cell” is a cell manifesting a cellproliferative disorder that is a precancer or a precancerous condition.A “cancer cell” or “cancerous cell” is a cell manifesting a cellproliferative disorder that is a cancer. Any reproducible means ofmeasurement may be used to identify cancer cells or precancerous cells.Cancer cells or precancerous cells can be identified by histologicaltyping or grading of a tissue sample (e.g., a biopsy sample). Cancercells or precancerous cells can be identified through the use ofappropriate molecular markers.

Exemplary non-cancerous conditions or disorders include, but are notlimited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; skin-related hyperproliferative disorders, psoriasis;eczema; atopic dermatitis; hyperpigmentation disorders, eye-relatedhyperproliferative disorders, age-related macular degeneration,ulcerative colitis; pancreatic fibrosis; hepatic fibrosis; acute andchronic renal disease; irritable bowel syndrome; pyresis; restenosis;cerebral malaria; stroke and ischemic injury; neural trauma; Alzheimer'sdisease; Huntington's disease; Parkinson's disease; acute and chronicpain; allergic rhinitis; allergic conjunctivitis; chronic heart failure;acute coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lymedisease; Reiter's syndrome; acute synovitis; muscle degeneration,bursitis; tendonitis; tenosynovitis; herniated, ruptures, or prolapsedintervertebral disk syndrome; osteopetrosis; thrombosis; restenosis;silicosis; pulmonary sarcosis; bone resorption diseases, such asosteoporosis; graft-versus-host reaction; fibroadipose hyperplasia;spinocerebullar ataxia type 1; CLOVES syndrome; Harlequin ichthyosis;macrodactyly syndrome; Proteus syndrome (Wiedemann syndrome); LEOPARDsyndrome; systemic sclerosis; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; diabetes mellitus;hemihyperplasia-multiple lipomatosis syndrome; megalencephaly; rarehypoglycemia, Klippel-Trenaunay syndrome; harmatoma; Cowden syndrome; orovergrowth-hyperglycemia.

Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, anal squamous cellcarcinoma, angiosarcoma, appendix cancer, childhood cerebellarastrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skincancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer,intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer,bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma,brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma,cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodeimal tumors, visual pathway andhypothalamic glioma, breast cancer, bronchial adenomas/carcinoids,carcinoid tumor, gastrointestinal, nervous system cancer, nervous systemlymphoma, central nervous system cancer, central nervous systemlymphoma, cervical cancer, childhood cancers, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma,lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrialcancer, esophageal cancer, extracranial germ cell tumor, extragonadalgerm cell tumor, extrahepatic bile duct cancer, eye cancer, intraocularmelanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, ovarian germ cell tumor, gestational trophoblastictumor glioma, head and neck cancer, head and neck squamous cellcarcinoma, hepatocellular (liver) cancer, Hodgkin lymphoma,hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet celltumors (endocrine pancreas), Kaposi Sarcoma, kidney cancer, renalcancer, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia,T-cell lymphoblastic leukemia, acute myeloid leukemia, chroniclymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia,lip and oral cavity cancer, liver cancer, lung cancer, non-small celllung cancer, small cell lung cancer, lung squamous cell carcinoma,AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervoussystem lymphoma, B-cell lymphoma, primary effusion lymphoma, Waldenstrammacroglobulinemia, medulloblastoma, melanoma, intraocular (eye)melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma,metastatic squamous neck cancer, mouth cancer, cancer of the tongue,multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,pancreatic endocrine tumor, paranasal sinus and nasal cavity cancer,parathyroid cancer, cholangiocarcinoma, penile cancer, pharyngealcancer, pheochromocytoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, pituitary adenoma, plasma cellneoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer,rectal cancer, renal pelvis and ureter, transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing family ofsarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterine cancer,uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma),merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma,squamous cell carcinoma, stomach (gastric) cancer, supratentorialprimitive neuroectodermal tumors, testicular cancer, throat cancer,thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cellcancer of the renal pelvis and ureter and other urinary organs,gestational trophoblastic tumor, urethral cancer, endometrial uterinecancer, uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvarcancer, and Wilm's Tumor.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. A cellproliferative disorder of the hematologic system can include lymphoma,leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benignmonoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoidpapulosis, polycythemia vera, chronic myelocytic leukemia, agnogenicmyeloid metaplasia, and essential thrombocythemia. A cell proliferativedisorder of the hematologic system can include hyperplasia, dysplasia,and metaplasia of cells of the hematologic system. Preferably,compositions of the present invention may be used to treat a cancerselected from the group consisting of a hematologic cancer of thepresent invention or a hematologic cell proliferative disorder of thepresent invention. A hematologic cancer of the present invention caninclude multiple myeloma, lymphoma (including Hodgkin's lymphoma,non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas oflymphocytic and cutaneous origin), leukemia (including childhoodleukemia, hairy-cell leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, chronic lymphocytic leukemia, chronic myelocyticleukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloidneoplasms and mast cell neoplasms.

A “cell proliferative disorder of the lung” is a cell proliferativedisorder involving cells of the lung. Cell proliferative disorders ofthe lung can include all forms of cell proliferative disorders affectinglung cells. Cell proliferative disorders of the lung can include lungcancer, a precancer or precancerous condition of the lung, benigngrowths or lesions of the lung, and malignant growths or lesions of thelung, and metastatic lesions in tissue and organs in the body other thanthe lung. Preferably, compositions of the present invention may be usedto treat lung cancer or cell proliferative disorders of the lung. Lungcancer can include all forms of cancer of the lung. Lung cancer caninclude malignant lung neoplasms, carcinoma in situ, typical carcinoidtumors, and atypical carcinoid tumors. Lung cancer can include smallcell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”),squamous cell carcinoma, adenocarcinoma, small cell carcinoma, largecell carcinoma, adenosquamous cell carcinoma, and mesothelioma. Lungcancer can include “scar carcinoma”, bronchioalveolar carcinoma, giantcell carcinoma, spindle cell carcinoma, and large cell neuroendocrinecarcinoma. Lung cancer can include lung neoplasms having histologic andultrastructual heterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can include all forms of cellproliferative disorders affecting lung cells. Cell proliferativedisorders of the lung can include lung cancer, precancerous conditionsof the lung. Cell proliferative disorders of the lung can includehyperplasia, metaplasia, and dysplasia of the lung. Cell proliferativedisorders of the lung can include asbestos-induced hyperplasia, squamousmetaplasia, and benign reactive mesothelial metaplasia. Cellproliferative disorders of the lung can include replacement of columnarepithelium with stratified squamous epithelium, and mucosal dysplasia.Individuals exposed to inhaled injurious environmental agents such ascigarette smoke and asbestos may be at increased risk for developingcell proliferative disorders of the lung. Prior lung diseases that maypredispose individuals to development of cell proliferative disorders ofthe lung can include chronic interstitial lung disease, necrotizingpulmonary disease, scleroderma, rheumatoid disease, sarcoidosis,interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathicpulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, andHodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. Preferably, the cellproliferative disorder of the colon is colon cancer. Preferably,compositions of the present invention may be used to treat colon canceror cell proliferative disorders of the colon. Colon cancer can includeall forms of cancer of the colon. Colon cancer can include sporadic andhereditary colon cancers. Colon cancer can include malignant colonneoplasms, carcinoma in situ, typical carcinoid tumors, and atypicalcarcinoid tumors. Colon cancer can include adenocarcinoma, squamous cellcarcinoma, and adenosquamous cell carcinoma. Colon cancer can beassociated with a hereditary syndrome selected from the group consistingof hereditary nonpolyposis colorectal cancer, familial adenomatouspolyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndromeand juvenile polyposis. Colon cancer can be caused by a hereditarysyndrome selected from the group consisting of hereditary nonpolyposiscolorectal cancer, familial adenomatous polyposis, Gardner's syndrome,Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.

Cell proliferative disorders of the colon can include all forms of cellproliferative disorders affecting colon cells. Cell proliferativedisorders of the colon can include colon cancer, precancerous conditionsof the colon, adenomatous polyps of the colon and metachronous lesionsof the colon. A cell proliferative disorder of the colon can includeadenoma. Cell proliferative disorders of the colon can be characterizedby hyperplasia, metaplasia, and dysplasia of the colon. Prior colondiseases that may predispose individuals to development of cellproliferative disorders of the colon can include prior colon cancer.Current disease that may predispose individuals to development of cellproliferative disorders of the colon can include Crohn's disease andulcerative colitis. A cell proliferative disorder of the colon can beassociated with a mutation in a gene selected from the group consistingof p53, ras, FAP and DCC. An individual can have an elevated risk ofdeveloping a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “cell proliferative disorder of the pancreas” is a cell proliferativedisorder involving cells of the pancreas. Cell proliferative disordersof the pancreas can include all forms of cell proliferative disordersaffecting pancreatic cells. Cell proliferative disorders of the pancreascan include pancreas cancer, a precancer or precancerous condition ofthe pancreas, hyperplasia of the pancreas, and dysaplasia of thepancreas, benign growths or lesions of the pancreas, and malignantgrowths or lesions of the pancreas, and metastatic lesions in tissue andorgans in the body other than the pancreas. Pancreatic cancer includesall forms of cancer of the pancreas. Pancreatic cancer can includeductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassifiedlarge cell carcinoma, small cell carcinoma, pancreatoblastoma, papillaryneoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serouscystadenoma. Pancreatic cancer can also include pancreatic neoplasmshaving histologic and ultrastructual heterogeneity (e.g., mixed celltypes).

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. Cell proliferative disordersof the prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, and malignantgrowths or lesions of the prostate, and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. Cell proliferative disorders ofthe skin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

A cell proliferative disorder of the breast can be a precancerouscondition of the breast. Compositions of the present invention may beused to treat a precancerous condition of the breast. A precancerouscondition of the breast can include atypical hyperplasia of the breast,ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer,or carcinoma in situ). A precancerous condition of the breast can bestaged according to the TNM classification scheme as accepted by theAmerican Joint Committee on Cancer (AJCC), where the primary tumor (T)has been assigned a stage of T0 or Tis; and where the regional lymphnodes (N) have been assigned a stage of N0; and where distant metastasis(M) has been assigned a stage of M0.

The cell proliferative disorder of the breast can be breast cancer.Preferably, compositions of the present invention may be used to treatbreast cancer. Breast cancer includes all forms of cancer of the breast.Breast cancer can include primary epithelial breast cancers. Breastcancer can include cancers in which the breast is involved by othertumors such as lymphoma, sarcoma or melanoma. Breast cancer can includecarcinoma of the breast, ductal carcinoma of the breast, lobularcarcinoma of the breast, undifferentiated carcinoma of the breast,cystosarcoma phyllodes of the breast, angiosarcoma of the breast, andprimary lymphoma of the breast. Breast cancer can include Stage I, II,IIIA, IIIB, IIIC and IV breast cancer. Ductal carcinoma of the breastcan include invasive carcinoma, invasive carcinoma in situ withpredominant intraductal component, inflammatory breast cancer, and aductal carcinoma of the breast with a histologic type selected from thegroup consisting of comedo, mucinous (colloid), medullary, medullarywith lymphcytic infiltrate, papillary, scirrhous, and tubular. Lobularcarcinoma of the breast can include invasive lobular carcinoma withpredominant in situ component, invasive lobular carcinoma, andinfiltrating lobular carcinoma. Breast cancer can include Paget'sdisease, extramammary Paget's disease, Paget's disease with intraductalcarcinoma, and Paget's disease with invasive ductal carcinoma. Breastcancer can include breast neoplasms having histologic and ultrastructualheterogeneity (e.g., mixed cell types). Breast cancer can be classifiedas a basal-like, luminal A, luminal B, ERBB2/Her2+ or normal breast-likemolecular subtype.

Preferably, compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, maybe used to treat breast cancer. A breast cancer that is to be treatedcan include familial breast cancer. A breast cancer that is to betreated can include sporadic breast cancer. A breast cancer that is tobe treated can arise in a male subject. A breast cancer that is to betreated can arise in a female subject. A breast cancer that is to betreated can arise in a premenopausal female subject or a postmenopausalfemale subject. A breast cancer that is to be treated can arise in asubject equal to or older than 30 years old, or a subject younger than30 years old. A breast cancer that is to be treated has arisen in asubject equal to or older than 50 years old, or a subject younger than50 years old. A breast cancer that is to be treated can arise in asubject equal to or older than 70 years old, or a subject younger than70 years old.

A breast cancer that is to be treated can be typed to identify afamilial or spontaneous mutation in BRCA1, BRCA2, or p53. A breastcancer that is to be treated can be typed as having a HER2/neu geneamplification, as overexpressing HER2/neu, or as having a low,intermediate or high level of HER2/neu expression. A breast cancer thatis to be treated can be typed for a marker selected from the groupconsisting of estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.A breast cancer that is to be treated can be typed as ER-unknown,ER-rich or ER-poor. A breast cancer that is to be treated can be typedas ER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. ER-typing of a breast cancer may beperformed as set forth in Onkologie 27: 175-179 (2004). A breast cancerthat is to be treated can be typed as PR-unknown, PR-rich or PR-poor. Abreast cancer that is to be treated can be typed as PR-negative orPR-positive. A breast cancer that is to be treated can be typed asreceptor positive or receptor negative. A breast cancer that is to betreated can be typed as being associated with elevated blood levels ofCA 15-3, or CA 27-29, or both.

A breast cancer that is to be treated can include a localized tumor ofthe breast. A breast cancer that is to be treated can include a tumor ofthe breast that is associated with a negative sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with a positive sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with one or more positive axillary lymphnodes, where the axillary lymph nodes have been staged by any applicablemethod. A breast cancer that is to be treated can include a tumor of thebreast that has been typed as having nodal negative status (e.g.,node-negative) or nodal positive status (e.g., node-positive). A breastcancer that is to be treated can include a tumor of the breast that hasmetastasized to other locations in the body. A breast cancer that is tobe treated can be classified as having metastasized to a locationselected from the group consisting of bone, lung, liver, or brain. Abreast cancer that is to be treated can be classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, may be used totreat or prevent a cell proliferative disorder of the breast, or totreat or prevent breast cancer, in a subject having an increased risk ofdeveloping breast cancer relative to the population at large. A subjectwith an increased risk of developing breast cancer relative to thepopulation at large is a female subject with a family history orpersonal history of breast cancer. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is a femalesubject having a germ-line or spontaneous mutation in BRCA1 or BRCA2, orboth. A subject with an increased risk of developing breast cancerrelative to the population at large is a female subject with a familyhistory of breast cancer and a germ-line or spontaneous mutation inBRCA1 or BRCA2, or both. A subject with an increased risk of developingbreast cancer relative to the population at large is a female who isgreater than 30 years old, greater than 40 years old, greater than 50years old, greater than 60 years old, greater than 70 years old, greaterthan 80 years old, or greater than 90 years old. A subject with anincreased risk of developing breast cancer relative to the population atlarge is a subject with atypical hyperplasia of the breast, ductalcarcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma insitu (LCIS), lobular neoplasia, or a stage 0 growth or lesion of thebreast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ).

A breast cancer that is to be treated can histologically gradedaccording to the Scarff-Bloom-Richardson system, wherein a breast tumorhas been assigned a mitosis count score of 1, 2, or 3; a nuclearpleiomorphism score of 1, 2, or 3; a tubule formation score of 1, 2, or3; and a total Scarff-Bloom-Richardson score of between 3 and 9. Abreast cancer that is to be treated can be assigned a tumor gradeaccording to the International Consensus Panel on the Treatment ofBreast Cancer selected from the group consisting of grade 1, grade 1-2,grade 2, grade 2-3, or grade 3.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2,T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pN0,PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1, PN1(mi), PN1a, PN1b,PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Acancer that is to be treated can be classified by microscopic appearanceas being associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder”. A normal cell lacks unregulatedor abnormal growth, or both, that can lead to the development of anunwanted condition or disease. Preferably, a normal cell possessesnormally functioning cell cycle checkpoint control mechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “candidate compound” refers to a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, that has been or will be tested in one ormore in vitro or in vivo biological assays, in order to determine ifthat compound is likely to elicit a desired biological or medicalresponse in a cell, tissue, system, animal or human that is being soughtby a researcher or clinician. A candidate compound is a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof. The biological or medicalresponse can be the treatment of cancer. The biological or medicalresponse can be treatment or prevention of a cell proliferativedisorder. In vitro or in vivo biological assays can include, but are notlimited to, enzymatic activity assays, electrophoretic mobility shiftassays, reporter gene assays, in vitro cell viability assays, and theassays described herein.

As used herein, “monotherapy” refers to the administration of a singleactive or therapeutic compound to a subject in need thereof. Preferably,monotherapy will involve administration of a therapeutically effectiveamount of an active compound. For example, cancer monotherapy with oneof the compounds of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, analog or derivative thereof, to asubject in need of treatment of cancer. Monotherapy may be contrastedwith combination therapy, in which a combination of multiple activecompounds is administered, preferably with each component of thecombination present in a therapeutically effective amount. In oneaspect, monotherapy with a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, is more effective than combination therapy in inducinga desired biological effect.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, to alleviate the symptoms or complicationsof a disease, condition or disorder, or to eliminate the disease,condition or disorder.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can also beused to prevent a disease, condition or disorder. As used herein,“preventing” or “prevent” describes reducing or eliminating the onset ofthe symptoms or complications of the disease, condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. In a preferred embodiment, the administration ofpharmaceutical compositions of the invention leads to the elimination ofa sign or symptom, however, elimination is not required. Effectivedosages are expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage, for example, according to the TNM system(accepted by the International Union Against Cancer (UICC) and theAmerican Joint Committee on Cancer (AJCC)) or by other art-recognizedmethods. Cancer stage refers to the extent or severity of the cancer,based on factors such as the location of the primary tumor, tumor size,number of tumors, and lymph node involvement (spread of cancer intolymph nodes). Alternatively, or in addition, severity is meant todescribe the tumor grade by art-recognized methods (see, National CancerInstitute, www.cancer.gov). Tumor grade is a system used to classifycancer cells in terms of how abnormal they look under a microscope andhow quickly the tumor is likely to grow and spread. Many factors areconsidered when determining tumor grade, including the structure andgrowth pattern of the cells. The specific factors used to determinetumor grade vary with each type of cancer. Severity also describes ahistologic grade, also called differentiation, which refers to how muchthe tumor cells resemble normal cells of the same tissue type (see,National Cancer Institute, www.cancer.gov). Furthermore, severitydescribes a nuclear grade, which refers to the size and shape of thenucleus in tumor cells and the percentage of tumor cells that aredividing (see, National Cancer Institute, www.cancer.gov).

In another aspect of the invention, severity describes the degree towhich a tumor has secreted growth factors, degraded the extracellularmatrix, become vascularized, lost adhesion to juxtaposed tissues, ormetastasized. Moreover, severity describes the number of locations towhich a primary tumor has metastasized. Finally, severity includes thedifficulty of treating tumors of varying types and locations. Forexample, inoperable tumors, those cancers which have greater access tomultiple body systems (hematological and immunological tumors), andthose which are the most resistant to traditional treatments areconsidered most severe. In these situations, prolonging the lifeexpectancy of the subject and/or reducing pain, decreasing theproportion of cancerous cells or restricting cells to one system, andimproving cancer stage/tumor grade/histological grade/nuclear grade areconsidered alleviating a sign or symptom of the cancer.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is not right in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined asnon-health-care professionals.

As used herein the term “sign” is also defined as an indication thatsomething is not right in the body. But signs are defined as things thatcan be seen by a doctor, nurse, or other health care professional.

Cancer is a group of diseases that may cause almost any sign or symptom.The signs and symptoms will depend on where the cancer is, the size ofthe cancer, and how much it affects the nearby organs or structures. Ifa cancer spreads (metastasizes), then symptoms may appear in differentparts of the body.

As a cancer grows, it begins to push on nearby organs, blood vessels,and nerves. This pressure creates some of the signs and symptoms ofcancer. If the cancer is in a critical area, such as certain parts ofthe brain, even the smallest tumor can cause early symptoms.

But sometimes cancers start in places where it does not cause anysymptoms until the cancer has grown quite large. Pancreas cancers, forexample, do not usually grow large enough to be felt from the outside ofthe body. Some pancreatic cancers do not cause symptoms until they beginto grow around nearby nerves (this causes a backache). Others growaround the bile duct, which blocks the flow of bile and leads to ayellowing of the skin known as jaundice. By the time a pancreatic cancercauses these signs or symptoms, it has usually reached an advancedstage.

A cancer may also cause symptoms such as fever, fatigue, or weight loss.This may be because cancer cells use up much of the body's energy supplyor release substances that change the body's metabolism. Or the cancermay cause the immune system to react in ways that produce thesesymptoms.

Sometimes, cancer cells release substances into the bloodstream thatcause symptoms not usually thought to result from cancers. For example,some cancers of the pancreas can release substances which cause bloodclots to develop in veins of the legs. Some lung cancers makehormone-like substances that affect blood calcium levels, affectingnerves and muscles and causing weakness and dizziness

Cancer presents several general signs or symptoms that occur when avariety of subtypes of cancer cells are present. Most people with cancerwill lose weight at some time with their disease. An unexplained(unintentional) weight loss of 10 pounds or more may be the first signof cancer, particularly cancers of the pancreas, stomach, esophagus, orlung.

Fever is very common with cancer, but is more often seen in advanceddisease. Almost all patients with cancer will have fever at some time,especially if the cancer or its treatment affects the immune system andmakes it harder for the body to fight infection. Less often, fever maybe an early sign of cancer, such as with leukemia or lymphoma.

Fatigue may be an important symptom as cancer progresses. It may happenearly, though, in cancers such as with leukemia, or if the cancer iscausing an ongoing loss of blood, as in some colon or stomach cancers.

Pain may be an early symptom with some cancers such as bone cancers ortesticular cancer. But most often pain is a symptom of advanced disease.

Along with cancers of the skin (see next section), some internal cancerscan cause skin signs that can be seen. These changes include the skinlooking darker (hyperpigmentation), yellow (jaundice), or red(erythema); itching; or excessive hair growth.

Alternatively, or in addition, cancer subtypes present specific signs orsymptoms. Changes in bowel habits or bladder function could indicatecancer. Long-term constipation, diarrhea, or a change in the size of thestool may be a sign of colon cancer. Pain with urination, blood in theurine, or a change in bladder function (such as more frequent or lessfrequent urination) could be related to bladder or prostate cancer.

Changes in skin condition or appearance of a new skin condition couldindicate cancer. Skin cancers may bleed and look like sores that do notheal. A long-lasting sore in the mouth could be an oral cancer,especially in patients who smoke, chew tobacco, or frequently drinkalcohol. Sores on the penis or vagina may either be signs of infectionor an early cancer.

Unusual bleeding or discharge could indicate cancer. Unusual bleedingcan happen in either early or advanced cancer. Blood in the sputum(phlegm) may be a sign of lung cancer. Blood in the stool (or a dark orblack stool) could be a sign of colon or rectal cancer. Cancer of thecervix or the endometrium (lining of the uterus) can cause vaginalbleeding. Blood in the urine may be a sign of bladder or kidney cancer.A bloody discharge from the nipple may be a sign of breast cancer.

A thickening or lump in the breast or in other parts of the body couldindicate the presence of a cancer. Many cancers can be felt through theskin, mostly in the breast, testicle, lymph nodes (glands), and the softtissues of the body. A lump or thickening may be an early or late signof cancer. Any lump or thickening could be indicative of cancer,especially if the formation is new or has grown in size.

Indigestion or trouble swallowing could indicate cancer. While thesesymptoms commonly have other causes, indigestion or swallowing problemsmay be a sign of cancer of the esophagus, stomach, or pharynx (throat).

Recent changes in a wart or mole could be indicative of cancer. Anywart, mole, or freckle that changes in color, size, or shape, or losesits definite borders indicates the potential development of cancer. Forexample, the skin lesion may be a melanoma.

A persistent cough or hoarseness could be indicative of cancer. A coughthat does not go away may be a sign of lung cancer. Hoarseness can be asign of cancer of the larynx (voice box) or thyroid.

While the signs and symptoms listed above are the more common ones seenwith cancer, there are many others that are less common and are notlisted here. However, all art-recognized signs and symptoms of cancerare contemplated and encompassed by the instant invention.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. A decrease in the mortality rateof a population may be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing initiation of treatment with an active compound. A decrease inthe mortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively on a canceror precancerous cell but not on a normal cell. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively to modulateone molecular target (e.g., a target kinase) but does not significantlymodulate another molecular target (e.g., a non-target kinase). Theinvention also provides a method for selectively inhibiting the activityof an enzyme, such as a kinase. Preferably, an event occurs selectivelyin population A relative to population B if it occurs greater than twotimes more frequently in population A as compared to population B. Anevent occurs selectively if it occurs greater than five times morefrequently in population A. An event occurs selectively if it occursgreater than ten times more frequently in population A; more preferably,greater than fifty times; even more preferably, greater than 100 times;and most preferably, greater than 1000 times more frequently inpopulation A as compared to population B. For example, cell death wouldbe said to occur selectively in cancer cells if it occurred greater thantwice as frequently in cancer cells as compared to normal cells.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can modulatethe activity of a molecular target (e.g., a target kinase). Modulatingrefers to stimulating or inhibiting an activity of a molecular target.Preferably, a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof,modulates the activity of a molecular target if it stimulates orinhibits the activity of the molecular target by at least 2-foldrelative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. Morepreferably, a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof,modulates the activity of a molecular target if it stimulates orinhibits the activity of the molecular target by at least 5-fold, atleast 10-fold, at least 20-fold, at least 50-fold, at least 100-foldrelative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. The activityof a molecular target may be measured by any reproducible means. Theactivity of a molecular target may be measured in vitro or in vivo. Forexample, the activity of a molecular target may be measured in vitro byan enzymatic activity assay or a DNA binding assay, or the activity of amolecular target may be measured in vivo by assaying for expression of areporter gene.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, does notsignificantly modulate the activity of a molecular target if theaddition of the compound does not stimulate or inhibit the activity ofthe molecular target by greater than 10% relative to the activity of themolecular target under the same conditions but lacking only the presenceof said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a kinase isozyme alpha in comparison to a kinase isozymebeta). Preferably, a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, demonstrates a minimum of a four fold differential,preferably a ten fold differential, more preferably a fifty folddifferential, in the dosage required to achieve a biological effect.Preferably, a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof,demonstrates this differential across the range of inhibition, and thedifferential is exemplified at the IC₅₀, i.e., a 50% inhibition, for amolecular target of interest.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof can result in modulation (i.e.,stimulation or inhibition) of an activity of a kinase of interest.

The present invention provides methods to assess biological activity ofa compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof. In one method,an assay based on enzymatic activity can be utilized. In one specificenzymatic activity assay, the enzymatic activity is from a kinase. Asused herein, “kinase” refers to a large class of enzymes which catalyzethe transfer of the γ-phosphate from ATP to the hydroxyl group on theside chain of Ser/Thr or Tyr in proteins and peptides and are intimatelyinvolved in the control of various important cell functions, perhapsmost notably: signal transduction, differentiation, and proliferation.There are estimated to be about 2,000 distinct protein kinases in thehuman body, and although each of these phosphorylates particularprotein/peptide substrates, they all bind the same second substrate ATPin a highly conserved pocket. About 50% of the known oncogene productsare protein tyrosine kinases (PTKs), and their kinase activity has beenshown to lead to cell transformation. Preferably, the kinase assayed isa tyrosine kinase.

A change in enzymatic activity caused by a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, can be measured in the disclosed assays.The change in enzymatic activity can be characterized by the change inthe extent of phosphorylation of certain substrates. As used herein,“phosphorylation” refers to the addition of phosphate groups to asubstrate, including proteins and organic molecules; and, plays animportant role in regulating the biological activities of proteins.Preferably, the phosphorylation assayed and measured involves theaddition of phosphate groups to tyrosine residues. The substrate can bea peptide or protein.

In some assays, immunological reagents, e.g., antibodies and antigens,are employed. Fluorescence can be utilized in the measurement ofenzymatic activity in some assays. As used herein, “fluorescence” refersto a process through which a molecule emits a photon as a result ofabsorbing an incoming photon of higher energy by the same molecule.Specific methods for assessing the biological activity of the disclosedcompounds are described in the examples.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof, to acell or a subject in need thereof results in modulation (i.e.,stimulation or inhibition) of an activity of an intracellular target(e.g., substrate). Several intracellular targets can be modulated withthe compounds of the present invention, including, but not limited to,adaptor proteins such as Gab-1, Grb-2, Shc, FRS2a, SHP2 and c-Cb1, andsignal transducers such as Ras, Src, PI3K, PLC-γ, STATs, ERK1 and 2 andFAK.

Activating refers to placing a composition of matter (e.g., protein ornucleic acid) in a state suitable for carrying out a desired biologicalfunction. A composition of matter capable of being activated also has anunactivated state. An activated composition of matter may have aninhibitory or stimulatory biological function, or both.

Elevation refers to an increase in a desired biological activity of acomposition of matter (e.g., a protein or a nucleic acid). Elevation mayoccur through an increase in concentration of a composition of matter.

As used herein, “a cell cycle checkpoint pathway” refers to abiochemical pathway that is involved in modulation of a cell cyclecheckpoint. A cell cycle checkpoint pathway may have stimulatory orinhibitory effects, or both, on one or more functions comprising a cellcycle checkpoint. A cell cycle checkpoint pathway is comprised of atleast two compositions of matter, preferably proteins, both of whichcontribute to modulation of a cell cycle checkpoint. A cell cyclecheckpoint pathway may be activated through an activation of one or moremembers of the cell cycle checkpoint pathway. Preferably, a cell cyclecheckpoint pathway is a biochemical signaling pathway.

As used herein, “cell cycle checkpoint regulator” refers to acomposition of matter that can function, at least in part, in modulationof a cell cycle checkpoint. A cell cycle checkpoint regulator may havestimulatory or inhibitory effects, or both, on one or more functionscomprising a cell cycle checkpoint. A cell cycle checkpoint regulatorcan be a protein or not a protein.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci USA . 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

Preferably, an effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, is not significantly cytotoxic to normal cells. Atherapeutically effective amount of a compound is not significantlycytotoxic to normal cells if administration of the compound in atherapeutically effective amount does not induce cell death in greaterthan 10% of normal cells. A therapeutically effective amount of acompound does not significantly affect the viability of normal cells ifadministration of the compound in a therapeutically effective amountdoes not induce cell death in greater than 10% of normal cells. In anaspect, cell death occurs by apoptosis.

Contacting a cell with a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, can induce or activate cell death selectively in cancercells. Administering to a subject in need thereof a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce or activate celldeath selectively in cancer cells. Contacting a cell with a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce cell deathselectively in one or more cells affected by a cell proliferativedisorder. Preferably, administering to a subject in need thereof acompound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, induces celldeath selectively in one or more cells affected by a cell proliferativedisorder.

The present invention relates to a method of treating or preventingcancer by administering a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, to a subject in need thereof, where administration ofthe compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, results in oneor more of the following: accumulation of cells in G1 and/or S phase ofthe cell cycle, cytotoxicity via cell death in cancer cells without asignificant amount of cell death in normal cells, antitumor activity inanimals with a therapeutic index of at least 2, and activation of a cellcycle checkpoint. As used herein, “therapeutic index” is the maximumtolerated dose divided by the efficacious dose.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention

As used herein, “combination therapy” or “co-therapy” includes theadministration of at least two compounds of the present invention, orpharmaceutically acceptable salts, prodrugs, metabolites, polymorphs orsolvates thereof, as part of a specific treatment regimen intended toprovide the beneficial effect from the co-action of these at least twocompounds of the present invention. The beneficial effect of thecombination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of these atleast two compounds of the present invention. Administration of these atleast two compounds of the present invention in combination typically iscarried out over a defined time period (usually minutes, hours, days orweeks depending upon the combination selected). “Combination therapy”may be, but generally is not, intended to encompass the administrationof two or more of these compounds of the present invention as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, wherein each therapeuticagent is administered at a different time, as well as administration ofthese therapeutic agents, or at least two of the therapeutic agents, ina substantially simultaneous manner. Substantially simultaneous manneras used herein is administration of the at least two therapeutic agentswithin 1 hour of each other. Substantially simultaneous administrationcan be accomplished, for example, by administering to the subject asingle composition having a fixed ratio of each therapeutic agent or inseparate capsules for each of the therapeutic agents. Sequential manneras used herein is administration of one of the at least two therapeuticagents more than one hour after the other of the at least twotherapeutic agents. Preferably, for sequential administration, one ofthe at least two therapeutic agents is administered at least 12 hours,at least 24 hours, at least 48 hours, at least 96 hours or at least oneweek after administration of the other therapeutic agent. Sequential orsubstantially simultaneous administration of each therapeutic agent canbe effected by any appropriate route including, but not limited to, oralroutes, intravenous routes, intramuscular routes, and direct absorptionthrough mucous membrane tissues. The therapeutic agents can beadministered by the same route or by different routes. For example, afirst therapeutic agent of the combination selected may be administeredby intravenous injection while the other therapeutic agents of thecombination may be administered orally. Alternatively, for example, alltherapeutic agents may be administered orally or all therapeutic agentsmay be administered by intravenous injection. The sequence in which thetherapeutic agents are administered is not narrowly critical.

“Combination therapy” also embraces the administration of the at leasttwo compounds of the present invention as described above in furthercombination with other biologically active ingredients and non-drugtherapies (e.g., surgery or radiation treatment). Where the combinationtherapy further comprises a non-drug treatment, the non-drug treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andnon-drug treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof, or acombination of at least two compounds of the present invention, orpharmaceutically acceptable salts, prodrugs, metabolites, polymorphs orsolvates thereof, may be further administered in combination with anadditional chemotherapeutic agent. The additional chemotherapeutic agent(also referred to as an anti-neoplastic agent or anti-proliferativeagent) can be an alkylating agent; an antibiotic; an anti-metabolite; adetoxifying agent; an interferon; a polyclonal or monoclonal antibody;an EGFR inhibitor; an FGFR inhibitor, a HER2 inhibitor; a histonedeacetylase inhibitor; a hormone; a mitotic inhibitor; an MTORinhibitor; a multi-kinase inhibitor; a serine/threonine kinaseinhibitor; a tyrosine kinase inhibitors; a VEGF/VEGFR inhibitor; ataxane or taxane derivative, an aromatase inhibitor, an anthracycline, amicrotubule targeting drug, a topoisomerase poison drug, an inhibitor ofa molecular target or enzyme (e.g., a kinase inhibitor), a cytidineanalogue drug or any chemotherapeutic, anti-neoplastic oranti-proliferative agent listed inwww.cancer.org/docroot/cdg/cdg_(—)0.asp.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary antibiotics include, but are not limited to, doxorubicin(Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone);bleomycin (Blenoxane); daunorubicin (Cerubidine); daunorubicin liposomal(DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin(Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin(Nipent); or valrubicin (Valstar).

Exemplary anti-metabolites include, but are not limited to, fluorouracil(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine(Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine(Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar);cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal(DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine(FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine(Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall);thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary detoxifying agents include, but are not limited to, amifostine(Ethyol) or mesna (Mesnex).

Exemplary interferons include, but are not limited to, interferonalfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are notlimited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab(Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab(Vectibix); tositumomab/iodine¹³¹ tositumomab (Bexxar); alemtuzumab(Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab(Mylotarg); eculizumab (Soliris) ordenosumab; nivolumab (Opdivo);pembrolizumab (Keytruda); ipilimumab (Yervoy); pidilizumab;atezolizumab.

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva);panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab(Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab(Herceptin); lapatinib (Tykerb) or AC-480.

Histone Deacetylase Inhibitors include, but are not limited to,vorinostat (Zolinza).

Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron;Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole(Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane(Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole(Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone(Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin);toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron);abarelix (Plenaxis); or testolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Exemplary MTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; orAP23573.

Exemplary multi-kinase inhibitors include, but are not limited to,sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474;PKC-412; motesanib; or AP24534.

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, ruboxistaurin; eril/easudil hydrochloride; flavopiridol;seliciclib (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc412;bryostatin; KAI-9803; SF1126; VX-680; Azd1152; Arry-142886 (AZD-6244);SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

Exemplary tyrosine kinase inhibitors include, but are not limited to,erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib(Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab(Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux);panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient);dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584);CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent);ranibizumab; pegaptanib; or vandetinib.

Exemplary microtubule targeting drugs include, but are not limited to,paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilonesand navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to,teniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to,paclitaxel and docetaxol.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexalen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak);porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid);bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel);arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine(Leucenol); (1M tegafur-0.4 M 5-chloro-2,4-dihydroxypyrimidine-1 Mpotassium oxonate) or lovastatin.

In another aspect, the additional chemotherapeutic agent can be acytokine such as G-CSF (granulocyte colony stimulating factor). Inanother aspect, a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, may be administered in combination with radiationtherapy. Radiation therapy can also be administered in combination witha compound of the present invention and another chemotherapeutic agentdescribed herein as part of a multiple agent therapy. In yet anotheraspect, a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, analog or derivative thereof, maybe administered in combination with standard chemotherapy combinationssuch as, but not restricted to, CMF (cyclophosphamide, methotrexate and5-fluorouracil), CAF (cyclophosphamide, adriamycin and 5-fluorouracil),AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin,and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, andpaclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP),Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a molarratio of 1:0.4:1), Camptothecin-11 (CPT-11, Irinotecan or Camptosar™) orCMFP (cyclophosphamide, methotrexate, 5-fluorouracil and prednisone).

In preferred embodiments, a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, may be administered with an inhibitor of an enzyme,such as a receptor or non-receptor kinase. Receptor and non-receptorkinases of the invention are, for example, tyrosine kinases orserine/threonine kinases. Kinase inhibitors of the invention are smallmolecules, polynucleic acids, polypeptides, or antibodies.

Exemplary kinase inhibitors include, but are not limited to, BIBW 2992(targets EGFR and Erb2), Cetuximab/Erbitux (targets Erb1),Imatinib/Gleevic (targets Bcr-Abl), Trastuzumab (targets Erb2),Gefitinib/Iressa (targets EGFR), Ranibizumab (targets VEGF), Pegaptanib(targets VEGF), Erlotinib/Tarceva (targets Erb1), Nilotinib (targetsBcr-Abl), Lapatinib (targets Erb1 and Erb2/Her2), GW-572016/lapatinibditosylate (targets HER2/Erb2), Panitumumab/Vectibix (targets EGFR),Vandetinib (targets RET/VEGFR), E7080 (multiple targets including RETand VEGFR), Herceptin (targets HER2/Erb2), PKI-166 (targets EGFR),Canertinib/CI-1033 (targets EGFR), Sunitinib/SU-11464/Sutent (targetsEGFR and FLT3), Matuzumab/Emd7200 (targets EGFR), EKB-569 (targetsEGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3),Vatalanib/Ptk787/ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614(targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322(targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606(targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154(targets JAK), WHI-P131 (targets JAK), sorafenib/Nexavar (targets RAFkinase, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, KIT, FLT-3, and RET),Dasatinib/Sprycel (BCR/ABL and Src), AC-220 (targets Flt3), AC-480(targets all HER proteins, “panHER”), Motesanib diphosphate (targetsVEGF1-3, PDGFR, and c-kit), Denosumab (targets RANKL, inhibits SRC),AMG888 (targets HER3), and AP24534 (multiple targets including Flt3).

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, Rapamune (targets mTOR/FRAP1), Deforolimus (targets mTOR),Certican/Everolimus (targets mTOR/FRAP1), AP23573 (targets mTOR/FRAP1),Eril/Fasudil hydrochloride (targets RHO), Flavopiridol (targets CDK),Seliciclib/CYC202/Roscovitrine (targets CDK), SNS-032/BMS-387032(targets CDK), Ruboxistaurin (targets PKC), Pkc412 (targets PKC),Bryostatin (targets PKC), KAI-9803 (targets PKC), SF1126 (targets PI3K),VX-680 (targets Aurora kinase), Azd1152 (targets Aurora kinase),Any-142886/AZD-6244 (targets MAP/MEK), SCID-469 (targets MAP/MEK),GW681323 (targets MAP/MEK), CC-401 (targets JNK), CEP-1347 (targetsJNK), and PD 332991 (targets CDK).

In particular embodiments, the compounds of the present invention(Compound 1, 2 or 3, or a pharmaceutically acceptable salt, solvate,hydrate, or prodrug thereof) can be combined with an FGFR or FGFR2inhibitor in the treatment of a cell proliferative disorder. In someembodiments, the FGFR or FGFR2 inhibitor is Compound 4, or apharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.In some embodiments, Compound 1 or 3, or a pharmaceutically acceptablesalt, solvate, hydrate, or prodrug thereof can be combined with Compound4, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof. In some embodiments, Compound 1, or a pharmaceuticallyacceptable salt, solvate, hydrate, or prodrug thereof) can be combinedwith Compound 4, or a pharmaceutically acceptable salt, solvate,hydrate, or prodrug thereof. In some embodiments, Compound 3, or apharmaceutically acceptable salt, solvate, hydrate, or prodrug thereofcan be combined with Compound 4, or a pharmaceutically acceptable salt,solvate, hydrate, or prodrug thereof. In some embodiments, Compound 2,or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof can be combined with Compound 4, or a pharmaceuticallyacceptable salt, solvate, hydrate, or prodrug thereof.

FGFR2 is a member of the fibroblast growth factor receptor family, whereamino acid sequence is highly conserved between members and throughoutevolution. FGFR family members differ from one another in their ligandaffinities and tissue distribution. A full-length representative proteinconsists of an extracellular region, composed of threeimmunoglobulin-like domains, a single hydrophobic membrane-spanningsegment and a cytoplasmic tyrosine kinase domain. The extracellularportion of the protein interacts with fibroblast growth factors, settingdownstream signals, ultimately influencing mitogenesis anddifferentiation.

Alterations in the activity (expression) of the FGFR2 gene areassociated with certain cancers. The altered gene expression may enhanceseveral cancer-related events such as cell proliferation, cell movement,and the development of new blood vessels that nourish a growing tumor.The FGFR2 gene is abnormally active (overexpressed) in certain types ofstomach cancers, and this amplification is associated with a poorerprognosis and response to standard clinical methods. Abnormal expressionof FGFR2 is also found in patients with prostate cancer. More than 60percent of women with breast cancer in the United States carry at leasta single mutation in this gene as well.

2. Compounds of the Present Invention

The present invention provides Compound 1, Compound 2 and Compound 3,synthetic methods for making these compounds, pharmaceuticalcompositions containing at least one of these compounds and various usesof the compounds.

Compound 1

(3-(3-(4-(1-aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine),or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof.

Compound 2

3-(3-(4-(1-aminocyclobutyl)phenyl)-5-(3-morpholinophenyl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine,or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof.

Compound 3

N-(1-(3-(3-(4-(1-aminocyclobutyl)phenyl)-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-5-yl)phenyl)piperidin-4-yl)-N-methylacetamide,or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof.

The present invention also provides Compound 4, synthetic methods formaking the compound, pharmaceutical compositions containing the compoundand various uses of the compound.

Compound 4

((R)-6-(2-fluorophenyl)-N-(3-(2-((2-methoxyethyl)amino)ethyl)phenyl)-5,6-dihydrobenzo[h]quinazolin-2-amine),or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof.

3. Definitions

As used herein, “alkyl”, “C₁, C₂, C₃, C₄, C₅ or C₆ alkyl” or “C₁-C₆alkyl” is intended to include C₁, C₂, C₃, C₄, C₅ or C₆ straight chain(linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. For example, C1-C6alkyl is intended to include C1, C2, C3, C4, C5 and C6 alkyl groups.Examples of alkyl include, moieties having from one to six carbon atoms,such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.

In certain embodiments, a straight chain or branched alkyl has six orfewer carbon atoms (e.g., C₁-C₆ for straight chain, C₃-C₆ for branchedchain), and in another embodiment, a straight chain or branched alkylhas four or fewer carbon atoms.

“Heteroalkyl” groups are alkyl groups, as defined above, that have anoxygen, nitrogen, sulfur or phosphorous atom replacing one or morehydrocarbon backbone carbon atoms.

As used herein, the term “cycloalkyl”, “C₃, C₄, C₅, C₆, C₇ or C₈cycloalkyl” or “C₃-C₈ cycloalkyl” is intended to include hydrocarbonrings having from three to eight carbon atoms in their ring structure.In one embodiment, a cycloalkyl group has five or six carbons in thering structure.

The term “substituted alkyl” refers to alkyl moieties havingsubstituents replacing one or more hydrogen atoms on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, amino (including alkylamino,dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)).

Unless the number of carbons is otherwise specified, “lower alkyl”includes an alkyl group, as defined above, having from one to six, or inanother embodiment from one to four, carbon atoms in its backbonestructure. “Lower alkenyl” and “lower alkynyl” have chain lengths of,for example, two to six or of two to four carbon atoms.

As used herein, “alkyl linker” is intended to include C₁, C₂, C₃, C₄, C₅or C₆ straight chain (linear) saturated aliphatic hydrocarbon groups andC₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbon groups. Forexample, C1-C6 alkyl linker is intended to include C1, C2, C3, C4, C5and C6 alkyl linker groups. Examples of alkyl linker include, moietieshaving from one to six carbon atoms, such as, but not limited to, methyl(—CH₂—), ethyl (—CH₂CH₂—), n-propyl (—CH₂CH₂CH₂—), i-propyl(—CHCH₃CH₂—), n-butyl (—CH₂CH₂CH₂CH₂—), s-butyl (—CHCH₃CH₂CH₂—), i-butyl(—C(CH₃)₂CH₂—), n-pentyl (—CH₂CH₂CH₂CH₂CH₂—), s-pentyl(—CHCH₃CH₂CH₂CH₂—) or n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₂—).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includes straightchain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl), branched alkenyl groups,cycloalkenyl (e.g., alicyclic) groups (e.g., cyclopropenyl,cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl oralkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenylsubstituted alkenyl groups. In certain embodiments, a straight chain orbranched alkenyl group has six or fewer carbon atoms in its backbone(e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). Likewise,cycloalkenyl groups may have from five to eight carbon atoms in theirring structure, and in one embodiment, cycloalkenyl groups have five orsix carbons in the ring structure. The term “C₂-C₆” includes alkenylgroups containing two to six carbon atoms. The term “C₃-C₆” includesalkenyl groups containing three to six carbon atoms.

“Heteroalkenyl” includes alkenyl groups, as defined herein, having anoxygen, nitrogen, sulfur or phosphorous atom replacing one or morehydrocarbon backbone carbons.

The term “substituted alkenyl” refers to alkenyl moieties havingsubstituents replacing one or more hydrogen atoms on one or morehydrocarbon backbone carbon atoms. Such substituents can include, forexample, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, amino (including alkylamino,dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic orheteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), branched alkynyl groups, andcycloalkyl or cycloalkenyl substituted alkynyl groups. In certainembodiments, a straight chain or branched alkynyl group has six or fewercarbon atoms in its backbone (e.g., C₂-C₆ for straight chain, C₃-C₆ forbranched chain). The term “C₂-C₆” includes alkynyl groups containing twoto six carbon atoms. The term “C₃-C₆” includes alkynyl groups containingthree to six carbon atoms.

“Heteroalkynyl” includes alkynyl groups, as defined herein, having anoxygen, nitrogen, sulfur or phosphorous atom replacing one or morehydrocarbon backbone carbons.

The term “substituted alkynyl” refers to alkynyl moieties havingsubstituents replacing one or more hydrogen atoms on one or morehydrocarbon backbone carbon atoms. Such substituents can include, forexample, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, amino (including alkylamino,dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aryl” includes groups with aromaticity, including “conjugated”, ormulticyclic, systems with at least one aromatic ring. Examples includephenyl, benzyl, etc.

“Heteroaryl” groups are aryl groups, as defined above, having from oneto four heteroatoms in the ring structure, and may also be referred toas “aryl heterocycles” or “heteroaromatics”. As used herein, the term“heteroaryl” is intended to include a stable 5-, 6-, or 7-memberedmonocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromaticheterocyclic ring which consists of carbon atoms and one or moreheteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms,independently selected from the group consisting of nitrogen, oxygen andsulfur. The nitrogen atom may be substituted or unsubstituted (i.e., Nor NR wherein R is H or other substituents, as defined). The nitrogenand sulfur heteroatoms may optionally be oxidized (i.e., N→O andS(O)_(p), where p=1 or 2). It is to be noted that total number of S andO atoms in the aromatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,naphthridine, indole, benzofuran, purine, benzofuran, deazapurine,indolizine.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

The aryl or heteroaryl aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,alkyl, alkenyl, akynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,phosphate, phosphonato, phosphinato, amino (including alkylamino,dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings, which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl).

As used herein, “carbocycle” or “carbocyclic ring” is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. For example, a C₃-C₁₄ carbocycle is intended to include amonocyclic, bicyclic or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 carbon atoms. Examples of carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl,cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl,adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl,naphthyl, indanyl, adamantyl and tetrahydronaphthyl. Bridged rings arealso included in the definition of carbocycle, including, for example,[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane and[2.2.2]bicyclooctane. A bridged ring occurs when one or more carbonatoms link two non-adjacent carbon atoms. In one embodiment, bridgerings are one or two carbon atoms. It is noted that a bridge alwaysconverts a monocyclic ring into a tricyclic ring. When a ring isbridged, the substituents recited for the ring may also be present onthe bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro ringsare also included.

As used herein, “heterocycle” includes any ring structure (saturated orpartially unsaturated) which contains at least one ring heteroatom(e.g., N, O or S). Examples of heterocycles include, but are not limitedto, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazineand tetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4H-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H, 6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

The term “substituted”, as used herein, means that any one or morehydrogen atmos on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogen atoms on the atomare replaced. Keto substituents are not present on aromatic moieties.Ring double bonds, as used herein, are double bonds that are formedbetween two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchformula. Combinations of substituents and/or variables are permissible,but only if such combinations result in stable compounds.

When any variable (e.g., R₁) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2 R₁ moieties,then the group may optionally be substituted with up to two R₁ moietiesand R₁ at each occurrence is selected independently from the definitionof R₁. Also, combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogen atoms are replaced by halogen atoms.

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties containing a carbonyl include, but are not limitedto, aldehydes, ketones, carboxylic acids, amides, esters, anhydrides,etc.

“Acyl” includes moieties that contain the acyl radical (—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, for example, alkyl groups,alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound toa carbonyl group. Examples of aroyl groups include phenylcarboxy,naphthyl carboxy, etc.

“Alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” include alkylgroups, as described above, wherein oxygen, nitrogen or sulfur atomsreplace one or more hydrocarbon backbone carbon atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms or heteroatoms. Forexample, the term includes “alkoxyalkyl”, which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to an alkyl group.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “thioalkyl” includes compounds or moieties which contain analkyl group connected with a sulfur atom. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes moieties which contain a sulfur atombonded to two carbon atoms or heteroatoms. Examples of thioethersinclude, but are not limited to alkthioalkyls, alkthioalkenyls andalkthioalkynyls. The term “alkthioalkyls” include moieties with analkyl, alkenyl or alkynyl group bonded to a sulfur atom which is bondedto an alkyl group. Similarly, the term “alkthioalkenyls” refers tomoieties wherein an alkyl, alkenyl or alkynyl group is bonded to asulfur atom which is covalently bonded to an alkenyl group; andalkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynylgroup is bonded to a sulfur atom which is covalently bonded to analkynyl group.

As used herein, “amine” or “amino” includes moieties where a nitrogenatom is covalently bonded to at least one carbon or heteroatom.“Alkylamino” includes groups of compounds wherein nitrogen is bound toat least one alkyl group. Examples of alkylamino groups includebenzylamino, methylamino, ethylamino, phenethylamino, etc.“Dialkylamino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups. Examples of dialkylamino groupsinclude, but are not limited to, dimethylamino and diethylamino“Arylamino” and “diarylamino” include groups wherein the nitrogen isbound to at least one or two aryl groups, respectively.“Alkylarylamino”, “alkylaminoaryl” or “arylaminoalkyl” refers to anamino group which is bound to at least one alkyl group and at least onearyl group. “Alkaminoalkyl” refers to an alkyl, alkenyl, or alkynylgroup bound to a nitrogen atom which is also bound to an alkyl group.“Acylamino” includes groups wherein nitrogen is bound to an acyl group.Examples of acylamino include, but are not limited to,alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom that is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups thatinclude alkyl, alkenyl or alkynyl groups bound to an amino group whichis bound to the carbon of a carbonyl or thiocarbonyl group. It alsoincludes “arylaminocarboxy” groups that include aryl or heteroarylmoieties bound to an amino group that is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”,“alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy”include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties,respectively, are bound to a nitrogen atom which is in turn bound to thecarbon of a carbonyl group. Amides can be substituted with substituentssuch as straight chain alkyl, branched alkyl, cycloalkyl, aryl,heteroaryl or heterocycle. Substituents on amide groups may be furthersubstituted.

Compounds of the present invention that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (m-CPBA) and/or hydrogen peroxides) to affordother compounds of the present invention. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O⁻). Furthermore, inother instances, the nitrogens in the compounds of the present inventioncan be converted to N-hydroxy or N-alkoxy compounds. For example,N-hydroxy compounds can be prepared by oxidation of the parent amine byan oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In addition, a crystal polymorphism may bepresent for the compounds represented by the formula. It is noted thatany crystal form, crystal form mixture, or anhydride or hydrate thereofis included in the scope of the present invention. Furthermore,so-called metabolite which is produced by degradation of the presentcompound in vivo is included in the scope of the present invention.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers”. Stereoisomers that are notmirror images of one another are termed “diastereoisomers”, andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture”.

A carbon atom bonded to four nonidentical substituents is termed a“chiral center”.

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture”. When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds. These configurations aredifferentiated in their names by the prefixes cis and trans, or Z and E,which indicate that the groups are on the same or opposite side of thedouble bond in the molecule according to the Cahn-Ingold-Prelog rules.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques; ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solid form,usually one tautomer predominates. In solutions where tautomerization ispossible, a chemical equilibrium of the tautomers will be reached. Theexact ratio of the tautomers depends on several factors, includingtemperature, solvent and pH. The concept of tautomers that areinterconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), amine-enamine andenamine-enamine.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofthe compounds does not exclude any tautomer form.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

4. Synthesis of Compounds of the Present Invention

Compounds of the present invention can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001; andGreene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999, incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentinvention.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the invention can tolerate a wide variety offunctional groups, therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof.

The present invention provides methods for the synthesis of Compound 1,Compound 2 and Compound 3. The present invention also provides detailedmethods for the synthesis of Compound 1, Compound 2 and Compound 3according to the following schemes and as shown in the examples.

Compound 1 may be prepared according to the following procedures fromcommercially available starting materials or starting materials whichcan be prepared using literature procedures. These procedures show thepreparation of Compound 1, 2 and 3.

General Procedure A

One general procedure for imidazo-pyridine formation is described belowin Scheme 1-1: Imidazo-pyridine formation

Step 1 Synthesis of 3-nitro-N-phenylpyridin-2-amine (Structure 3 asShown in Scheme 1-1)

2-chloro-3-nitropyridine 1 was dissolved in dioxane (10 mL/mmol) in around bottom flask. Aniline (structure 2 as shown in Scheme 1-1) wasadded (1.1 eq.) and diisopropylethylamine (3 eq.). The reaction mixturewas heated to the appropriate temperature for 4 to 36 hours. Aftercooling to room temperature the solvent was removed under reducedpressure. The residue was dissolved in ethyl acetate (20 mL/mmol) andwashed with water and brine (20 mL/mmol respectively). The organic phasewas separated and dried over Na₂SO₄. After filtration the solvent wasremoved under reduced pressure. The crude product (red to brown solid)was carried on to the next step without further purification.

Step 2 Synthesis of3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (Structure 5as Shown in Scheme 1-1)

3-nitro-N-phenylpyridin-2-amine (structure 3 as shown in Scheme 1-1) wasdissolved in dimethylsulfoxide (8 mL/mmol) and methanol (1.5 mL/mmol) ina round bottom flask. 2-aminonicotinaldehyde (structure 4 as shown inScheme 1-1) (1.1 eq.) was added and Na₂S₂O₄ (85%, 2.5 eq.). The reactionmixture was heated to 100° C. for 15 to 36 hours. After cooling to roomtemperature the reaction mixture was diluted with dichloromethane (20mL/mmol) and washed with water and brine. The organic phase wasseparated and dried over Na₂SO₄. After filtration the solvent wasremoved under reduced pressure. The crude product was purified by silicagel chromatography (dichloromethane/methanol; 0-20% methanol over 60min) to give a yellow to brown solid.

General Procedure A-1

One general procedure for R₂-amino-substituted imidazopyridine formationis described below in Scheme 1-2: Imidazol pyridine formation with aminosubstitution on pyridine

Step 1 Synthesis of 3-nitro-N-phenylpyridin-2-amine (Structure 3 asShown in Scheme 1-2)

2-chloro-3-nitropyridine (structure 1 as shown in Scheme 1-2) wasdissolved in dioxane (10 mL/mmol) in a round bottom flask. Aniline(structure 2 as shown in Scheme 1-2) was added (1.1 eq.) anddiisopropylethylamine (3 eq.). The reaction mixture was heated to theappropriate temperature for 4 to 36 hours. After cooling to roomtemperature the solvent was removed under reduced pressure. The residuewas dissolved in ethyl acetate (20 mL/mmol) and washed with water andbrine (20 mL/mmol respectively). The organic phase was separated anddried over Na₂SO₄. After filtration the solvent was removed underreduced pressure. The crude product (red to brown solid) was carried onto the next step without further purification.

Step 1-1 Synthesis ofN¹-alkyl/aryl-3-nitro-N²-phenylpyridine-2,6-diamine (Structure 3b asShown in Scheme 1-2)

Intermediate (structure 3a as shown in Scheme 1-2) (1 eq.) was dissolvedin dioxane (5 mL/mmol) in a round bottom flask. Alky/arylamine (2 eq.)was added and diisopropylamine (2.5 eq.). The reaction mixture washeated to 80° C. in an oil bath for 24 h. After cooling to roomtemperature the solvent was removed under reduced pressure. The residuewas dissolved in ethyl acetate (10 mL/mmol) and washed with water andbrine (5 mL/mmol respectively). The organic phase was separated anddried over Na₂SO₄. After filtration the solvent was removed underreduced pressure. The crude product (structure 3b as shown in Scheme1-2) was carried on to the next step without further purification.

Step 2 Synthesis of3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (Structure 5as Shown in Scheme 1-2)

3-nitro-N-phenylpyridin-2-amine (structure 3 as shown in Scheme 1-2) wasdissolved in dimethylsulfoxide (8 mL/mmol) and methanol (1.5 mL/mmol) ina round bottom flask. 2-aminonicotinaldehyde 4 (1.1 eq.) was added andNa₂S₂O₄ (85%, 2.5 eq.). The reaction mixture was heated to 100° C. for15 to 36 hours. After cooling to room temperature the reaction mixturewas diluted with dichloromethane (20 mL/mmol) and washed with water andbrine. The organic phase was separated and dried over Na₂SO₄. Afterfiltration the solvent was removed under reduced pressure. The crudeproduct was purified by silica gel chromatography(dichloromethane/methanol; 0-20% methanol over 60 min) to give a yellowto brown solid.

General Procedure B

One general procedure for BOC group deprotection is described below inScheme 2: Deprotection of BOC-group

Carbamate (structure 6 as shown in Scheme 2) (1 eq.) was dissolved inmethanol. HCl (20 eq., 4 M in dioxane) was added and stirred at roomtemperature for 2 to 4 hours. Concentration of the solution underreduced pressure gave the deprotected amine (structure 7 as shown inScheme 2) as hydrochloric acid salt, which was used for the next stepwithout further purification.

General Procedure C

The general procedures for Suzuki couplings are described below inscheme 8 and scheme 9.

Organo halide (structure 22 as shown in Scheme 8) (1 eq.), CsCO₃ (1eq.), Pd(PPh₃)₄ (0.1 eq.) and aryl boronic acid (2 eq.) were dissolvedin DMF. After degassing with nitrogen for 10 min the reaction mixturewas heated in the microwave for 15 min to 150° C. The reaction mixturewas filtered through a Bakerbound filtration column and purified byreverse phase preparative HPLC (water 0.05 M TFA/ACN 0.05M TFA 0-100%ACN) without prior removal of the solvent or the solvent was removedunder reduced pressure and the crude product (structure 14 as shown inScheme 8) was purified by silica gel chromatography (0-20% methanol indichloromethane).

General Procedure D

Organo halide (structure 15 as shown in Scheme 9) (1 eq.) was suspendedin a mixture of ethanol and toluene, 10 mL/mmol respectively. A solutionof NaHCO₃ sat. was added (3 mL/mmol). The reaction mixture was degassedwith nitrogen for 30 min. Subsequently it was heated to 100° C.overnight under nitrogen. After cooling down to room temperature it wasdiluted with dichloromethane (20 mL/mmol) and water (10 mL/mmol). Theorganic phase was separated and washed with brine (10 mL/mmol) and driedover Na₂SO₄. After filtration the solvent was removed in vacuo. Thecrude residue (structure 16 as shown in Scheme 9) was purified by silicagel chromatography (3-20% methanol in ethyl acetate).

General Procedure E

Intermediate (structure 19 as shown in Scheme 11) (1 eq.) was dissolvedin tetrahydrofuran, 10 mL/mmol Under inert atmosphere conditions, thealkyl/aryl zinc halide (1.5 eq.), Pd(PtBu₃)₂ (0.1 eq.) and potassiumtert-butoxide (1 eq.) were added. The reaction mixture was degassed withnitrogen for 30 min. Subsequently it was heated to 100° C. for 15 min inthe microwave. After cooling down to room temperature the reactionmixture was filtered through celite. It was diluted with dichloromethane(20 mL/mmol) and water (20 mL/mmol). Ethylenediamine tetraacetic acid(EDTA) (1 eq.) was added. The organic phase was separated and washedwith brine (10 mL/mmol) and dried over Na₂SO₄. After filtration thesolvent was removed in under reduced pressure. The crude residue 20 waspurified by silica gel chromatography (3-20% methanol in ethyl acetate).

Synthesis of Substituted 5,6-Dihydro-6-Phenylbenzo[f]Isoquinolin-2-AmineCompound

The present invention provides methods for the synthesis of Compound 4.The present invention also provides detailed methods for the synthesisof Compound 4 according to the following schemes as shown in theExamples.

Compound 4 may be prepared according to the following procedures fromcommercially available starting materials or starting materials whichcan be prepared using literature procedures. These procedures show thepreparation of Compound 4.

General Procedure 1

Step 1 Guanidine Formation

A 1 M solution of DIPEA in anhydrous DMF is prepared (solution A). A 0.5M solution of 1-H-pyrazole-1-carboxamidine hydrochloride is preparedusing solution A. A 0.25 M solutions of amines in anhydrous DMF is alsoprepared. Dispense 800 μL (200 μmol, 1.0 eq) of amine solution to 2-dramvials. Dispense 400 μL (200 μmol, 1.0 eq) of1-H-pyrazole-1-carboxamidine hydrochloride solution to vials. Dispenseneat 80 μL (2.3 eq) of DIPEA. Cap and vortex vials. Shake at 100° C. for12-24 hours. Look for disappearance of starting amine Continue heatingif amine is still present. Evaporate solvent until dry/oily. Anyremaining moisture was removed by doing Azeotrope with dry acetone (1mL), then evaporating again.

Step 2 Cyclization (Pyrimidine Formation)

Prepare 0.1 M solution of either(E)-2-((dimethylamino)methylene)-4-phenyl-3,4-dihydronaphthalen-1(2H)-oneor(E)-4-(3,4-dichlorophenyl)-2-((dimethylamino)methylene)-3,4-dihydronaphthalen-1(2H)-onein 200 proof EtOH. Dispense 2000 μL of EtOH to the residue from theprevious step. Dispense 2000 μL (200 μmol, 1.0 eq) of either(E)-2-((dimethylamino)methylene)-4-phenyl-3,4-dihydronaphthalen-1(2H)-oneor(E)-4-(3,4-dichlorophenyl)-2-((dimethylamino)methylene)-3,4-dihydronaphthalen-1(2H)-oneto the residue from the step 1. Dispense a solution of sodium ethoxidein ethanol (Aldrich, 21% by weight) to each vial 75 μL, 200 μmol. Shakeat 80° C. for 72 hours. Evaporate solvent until dry/oily. Dispense 2000μL water and 2000 μL of ethyl acetate. Let shake at 70° C. for 1 hour todissolve. Transfer 1200 μL of top organic layer to new vials. Dispense2000 μL ethyl acetate. Transfer 2300 μL of top organic layer to newvials. Evaporate the combined organics to dryness and samples werepurified by reverse phase chromatography on a preparative LC/UV/MSsystem using a mass triggered fractionation. Compounds were eluted fromthe HPLC column (Maccel 120-10-C18 SH 10 μm 20 mmID×50 mm) at 88 ml/minwith acetonitrile/water gradient using 0.1% TFA as modifier.

General Procedure 2

Compounds of the present invention can also be conveniently prepared bythe general procedure shown below.

Step 1(R)-2-((Dimethylamino)methylene)-4-(2-fluorophenyl)-3,4-dihydronaphthalen-1(2H)-one

A solution of (R)-4-(2-fluorophenyl)-3,4-dihydronaphthalen-1(2H)-one(8.0 g, 33.33 mmol) in N,N-dimethylformamide dimethylacetal (80 mL) washeated at 100° C. for 40 h. After the reaction mixture was cooled toroom temperature, hexane (50 mL) was added. Product was collected byfiltration and dried under high vacuum overnight to yield the titlecompound as yellow needles (6.95 g, 70% yield). ¹H-NMR (DMSO-d₆) δ 7.92(dd, J=7.2 and 1.6 Hz, 1H), 7.57 (s, 1H), 7.42-7.34 (m, 2H), 7.31-7.21(m, 2H), 7.09 (t, 1H), 6.91-6.88 (m, 2H), 4.48 (t, J=7.2 Hz, 1H),3.25-3.13 (m, 2H), 3.02 (s, 6H). LCMS m/e 296 [M+H].

Step 2:(R)-2-(3-(6-(2-Fluorophenyl)-5,6-dihydrobenzo[h]quinazolin-2-ylamino)phenyl)ethanol

To a mixture of(R)-2-((dimethylamino)methylene)-4-(2-fluorophenyl)-3,4-dihydronaphthalen-1(2H)-one(4.20 g, 14.24 mmol) and 1-(3-(2-hydroxyethyl)phenyl)guanidinehydrochloride salt (6.17 g, 28.47 mmol) in ethanol (40 mL) was addedsodium ethoxide (21% w/w in ethanol) (9.60 mL, 25.62 mmol). The mixturewas heated at 80° C. for 24 h and filtered while it was still hot. Solidwas washed with acetone (50 mL). Filtrate was concentrated to dryness toyield the crude product. The crude product was dissolved in ethanol (20mL) at 80° C. The product was precipitated after cooling down to roomtemperature over 2 hours. Solid was collected by filtration and thendissolved in acetone (30 mL) in another flask. To this acetone solutionwas added slowly 120 mL of water, and the resulting suspension wasstirred at room temperature for 30 min. and filtered. The solid wasdried at 50° C. under high vacuum for 24 hours to yield the titlecompound as a yellow solid (3.78 g, 65% yield). ¹H-NMR (DMSO-d₆) δ 9.52(s, 1H), 8.38-8.36 (dd, 1H), 8.32 (s, 1H), 7.74 (s, 1H), 7.68 (d, J=9.2Hz, 1H), 7.54-7.45 (m, 2H), 7.32-7.20 (m, 3H), 7.07-7.02 (m, 2H),6.83-6.78 (m, 2H), 4.72-4.65 (m, 2H), 3.68-3.63 (m, 2H), 3.22-3.07 (m,2H), 2.73 (t, J=7.6 Hz, 2H). LCMS m/e 412 [M+H].

Step 3(R)-3-(6-(2-Fluorophenyl)-5,6-dihydrobenzo[h]quinazolin-2-ylamino)phenethylmethanesulfonate

To a solution of(R)-2-(3-(6-(2-fluorophenyl)-5,6-dihydrobenzo[h]quinazolin-2-ylamino)phenyl)ethanol(4.59 g, 11.17 mmol) in dichloromethane (50 mL) was added triethylamine(2.33 mL, 16.75 mmol) and methanesulfonyl chloride (0.95 mL, 12.28mmol). The mixture was stirred at room temperature for 1 h, washed withwater (60 mL×3), dried over sodium sulfate and concentrated to yield thetitle compound as a yellow solid (5.37 g, 98% yield). ¹H-NMR (DMSO-d₆) δ9.59 (s, 1H), 8.37-8.35 (dd, 1H), 8.33 (s, 1H), 7.80 (s, 1H), 7.71 (d,J=10.4 Hz, 1H), 7.54-7.45 (m, 2H), 7.29-7.22 (m, 3H), 7.07-7.02 (m, 2H),6.82-6.78 (m, 2H), 4.67 (t, J=6.8 Hz, 1H), 4.45 (t, J=6.8 Hz, 2H),3.22-3.08 (m, 2H), 3.13 (s, 3H), 3.01 (t, J=6.4 Hz, 2H). LCMS m/e 490[M+H].

Step 4(R)-6-(2-Fluorophenyl)-N-(3-(2-(4-(2-methoxyethyl)piperazin-1-yl)ethyl)phenyl)-5,6-dihydrobenzo[h]quinazolin-2-amine

A solution of(R)-3-(6-(2-fluorophenyl)-5,6-dihydrobenzo[h]quinazolin-2-ylamino)phenethylmethanesulfonate (5.37 g, 11.00 mmol), 1-(2-methoxyethyl)piperazine(3.32 mL, 22.34 mmol) and triethylamine (1.5 mL, 11.17 mmol) inN,N-dimethylacetamide (30 mL) was heated at 90° C. for 20 h. Aftercooling to room temperature, water (200 mL) was added while stirring.The suspension was stirred for 15 min. and filtered. Solid was takeninto dichloromethane (200 mL), dried over sodium sulfate andconcentrated. Product was purified by flash column chromatography onsilica gel (120 g silica gel column, 0-10% 7N NH₃ inmethanol-dichloromethane, over 80 min.) to afford the title compound asa yellow solid (5.50 g, 93% yield). ¹H-NMR (DMSO-d₆): δ 9.53 (s, 1H),8.37-8.34 (m, 1H), 8.33 (s, 1H), 7.81 (s, 1H), 7.62-7.60 (m, 1H),7.51-7.46 (m, 2H), 7.30-7.19 (m, 3H), 7.08-7.02 (m, 2H), 6.82-6.79 (m,2H), 4.67 (t, J=7.2 Hz, 1H), 3.41 (t, J=5.6 Hz, 2H), 3.22-3.08 (m, 2H),3.33 (s, 3H), 2.74-2.70 (m, 2H), 2.59-2.42 (m, 12H). LCMS m/e 539 [M+H].

Step 5(R)-6-(2-Fluorophenyl)-N-(3-(2-(4-(2-methoxyethyl)piperazin-1-yl)ethyl)phenyl)-5,6-dihydrobenzo[h]quinazolin-2-aminehydrochloride salt

A solution of(R)-6-(2-fluorophenyl)-N-(3-(2-(4-(2-methoxyethyl)piperazin-1-yl)ethyl)phenyl)-5,6-dihydrobenzo[h]quinazolin-2-amine(5.5 g, 10.22 mmol) was dissolved in a mixed solvents of dichloromethane(30 mL) and ethyl acetate (20 mL). To this solution was added 2.5 M HClin ethyl acetate (30 mL) slowly while stirring. After addition, thesuspension was stirred at room temperature for 10 min, and then diethylether (300 mL) was added. Product was collected by filtration and driedat 60° C. for 24 hours to provide 6.2 g (˜93%) of final product as ayellow solid. The purity of this salt was found to be 100% at UV 254 nmby HPLC short method (2.5 min run) and 92% at UV254 by HPLC long method(20 min run). The salt was further purified as shown in the examplesdisclosed herein.

5. Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising at least one compound described herein in combination with atleast one pharmaceutically acceptable excipient or carrier.

A “pharmaceutical composition” is a formulation containing the compoundsof the present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A compound or pharmaceutical composition of the invention can beadministered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for treatment ofcancers, a compound of the invention may be injected directly intotumors, injected into the blood stream or body cavities or taken orallyor applied through the skin with patches. The dose chosen should besufficient to constitute effective treatment but not as high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, precancer, and the like) and the health of the patient shouldpreferably be closely monitored during and for a reasonable period aftertreatment.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to be treated is cancer. In another aspect, thedisease or condition to be treated is a cell proliferative disorder.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the invention vary depending on theagent, the age, weight, and clinical condition of the recipient patient,and the experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Generally, the dose should be sufficient to result in slowing,and preferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. Dosages can range from about0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects,dosages can range from about 1 mg/kg per day to about 1000 mg/kg perday. In an aspect, the dose will be in the range of about 0.1 mg/day toabout 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day toabout 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about1 g/day, in single, divided, or continuous doses (which dose may beadjusted for the patient's weight in kg, body surface area in m², andage in years). An effective amount of a pharmaceutical agent is thatwhich provides an objectively identifiable improvement as noted by theclinician or other qualified observer. For example, regression of atumor in a patient may be measured with reference to the diameter of atumor. Decrease in the diameter of a tumor indicates regression.Regression is also indicated by failure of tumors to reoccur aftertreatment has stopped. As used herein, the term “dosage effectivemanner” refers to amount of an active compound to produce the desiredbiological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention are capable of further formingsalts. All of these forms are also contemplated within the scope of theclaimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds of the present invention can also be prepared as esters,for example, pharmaceutically acceptable esters. For example, acarboxylic acid function group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., an acetate, propionate or other ester.

The compounds of the present invention can also be prepared as prodrugs,for example, pharmaceutically acceptable prodrugs. The terms “pro-drug”and “prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs in thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxy, amino,sulfhydryl, carboxy or carbonyl group is bonded to any group that may becleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxy or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention, and the like, See Bundegaard, H., Design of Prodrugs, p 1-92,Elesevier, New York-Oxford (1985).

The compounds, or pharmaceutically acceptable salts, esters or prodrugsthereof, are administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

The dosage regimen can be daily administration (e.g. every 24 hours) ofa compound of the present invention. The dosage regimen can be dailyadministration for consecutive days, for example, at least two, at leastthree, at least four, at least five, at least six or at least sevenconsecutive days. Dosing can be more than one time daily, for example,twice, three times or four times daily (per a 24 hour period). Thedosing regimen can be a daily administration followed by at least oneday, at least two days, at least three days, at least four days, atleast five days, or at least six days, without administration. Forexample, a compound of the present invention is administered at leastonce in a 24 hour period, then a compound of the present invention isnot administered for at least six days, then a compound of the presentinvention is administered to a subject in need.

The dosage regimen can include administering daily for at least oneweek, at least two weeks, or at least three weeks. Preferably, thedosage regimen can include administering about 50 mg to about 100 mgdaily. More preferably, the dosage regimen can include administeringabout 60 mg daily.

The dosage regimen can include administering once a week. Specifically,administering once during a week period. More specifically, thecomposition is administered at least once in 24 hours, not administeredfor at least six days, and administered at least once in 24 hoursfollowing the at least six days. Preferably, the dosage regimen caninclude administering about 250 mg to about 250 mg one day per week.More preferably, the dosage regimine can include administering about 300mg one day per week.

The dosage regimen can include administering daily for at least a week,ceasing administration for at least a week, and then administering dailyfor at least another week. For example, a compound of the presentinvention is administered daily for at least a week, no compounds of thepresent invention are administered for a second week, then a compound ofthe present invention is administered daily for at least a third week.Preferably, the dosage regimen can include administering about 150 mg toabout 250 mg daily. More preferably, the dosage regimen can includeadministering about 200 mg daily.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

6. Examples Example 1 Synthesis of3-(3-(4-(1-aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine(Compound 1) hydrochloride

3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-aminehydrochloride was synthesized according to General Procedure A followedby General Procedures D and B.

Step 1: tert-butyl(1-(4-((6-chloro-3-nitropyridin-2-yl)amino)phenyl)cyclobutyl) carbamate

To a solution of 2,6-dichloro-3-nitropyridine (5.11 g) in DMA (50 ml)and triethylamine (5 ml) chilled to 0° C. was added drop-wise a solutionof tert-butyl (1-(4-aminophenyl) cyclobutyl)carbamate (6.3 g) in DMA (25ml) over the course of 20 minutes. The reaction was allowed to stir at0° C. of one hour and then slowly allowed to warm to room temperatureand react overnight. Upon completion, the reaction was diluted withwater (250 mL) and extracted with ethyl acetate (2×200 ml). The organicswere combined, washed with saturated sodium bicarbonate solution (1×200ml), water (1×200 ml) and brine (1×100 ml). The organics were dried oversodium sulfate and concentrated under reduced pressure. Purification bycolumn chromatography (15% ethyl acetate in hexanes) gave the product asan orange solid (5.05 g, 50%). 400 M Hz ¹H-NMR (DMSO-d₆) δ: 10.05 (s,1H), 8.52 (d, J=8.8 Hz, 1H), 7.56-7.52 (m, 2H), 7.42-7.37 (m, 3H), 6.98(d, J=8.8 Hz, 1H), 2.47-2.34 (m, 4H), 2.04-1.96 (m, 1H), 1.84-1.74 (m,1H), 1.30 (bs, 9H); LCMS: 419 [M+H].

Step 2 tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate

To a solution of tert-butyl(1-(4-((6-chloro-3-nitropyridin-2-yl)amino)phenyl)cyclobutyl) carbamate(5.0 g) in anhydrous DMSO (60 ml) and anhydrous methanol (10 ml) wasadded 2-aminonicotinaldehyde (1.53 g) followed by Na₂S₂O₄ (6.25 g). Thereaction mixture was heated to 100° C. for 2 days. Upon completion ofthe reaction, water (250 ml) was added and the reaction was allowed tostir for 1 day at room temperature. The reaction was extracted withdichloromethane (2×200 ml). Upon extracting the second time, a largeamount of yellow solid precipitated from the water layer and the organiclayer. The solid was filtered off and found to be product. The productwas combined with the organic layers and dried under reduced pressure,giving the product as a yellow solid (3.1 g, 52%). 400 M Hz ¹H NMR(DMSO-d₆) δ: 8.26 (d, J=8.0 Hz, 1H), 8.00-7.96 (m, 1H), 7.69 (bs, 1H),7.54-7.35 (m, 5H), 7.24-7.08 (m, 1H), 7.04-6.96 (m, 2H), 6.32-6.28 (m,1H), 2.48-2.35 (m, 4H), 2.06-1.96 (m, 1H), 1.86-1.76 (m, 1H), 1.40-1.06(m, 9H); LCMS: 491 [M+H].

Step 3 tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-phenyl-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate

To a suspension of tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate(20 g) in toluene (200 mL) and ethanol (200 mL) was added saturatedaqueous sodium bicarbonate (150 mL) and phenyl boronic acid (9.9 g). Thereaction was degassed for 5 minutes and the Pd (PPh₃)₄ (1.0 g) wasadded. The reaction was again degassed for 5 minutes and then heated to100° C. for 2 days or until reaction is complete by LCMS. The reactionmixture was cooled to room temperature, and dichloromethane (250 ml×3)and water (100 mL) were added to the reaction. The organics were washedwith saturated sodium bicarbonate (1×250 mL) and water (1×250 mL), driedover sodium sulfate and concentrated. Purification by columnchromatography (10-100% ethyl acetate in hexanes) gave the product withsome impurities. The solid was re-crystallized with ethyl acetateaffording an off-white solid (7.2 g). 400 M Hz ¹H NMR (DMSO-d₆) δ: 8.23(d, J=8.0 Hz, 1H), 8.04-7.98 (m, 3H), 7.94 (d, J=8.0 Hz, 1H), 7.55 (d,J=8.8 Hz, 2H), 7.46-7.35 (m, 6H), 7.18-7.14 (m, 1H), 6.90 (bs, 1H), 6.33(dd, J=7.6 Hz and 4.4 Hz, 1H), 2.48-2.40 (m, 4H), 2.09-2.00 (m, 1H),1.89-1.79 (m, 1H), 1.30 (m, 9H); LCMS: 533 [M+H].

Step 43-(3-(4-(1-aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-aminehydrochloride

To a solution of tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-phenyl-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate(4.1 g) in dichloromethane (100 mL) was slowly added 4.0 M HCl indioxane (20 mL). The reaction was allowed to stir at room temperaturefor 2.5 hours. Upon completion of the reaction, ether (50 mL) was addedto the suspension and the solid was filtered to give product (4.032 g)as a white solid. ¹H NMR (DMSO-d₆) 400 MHz δ: 8.94 (s, 3H), 8.47 (bs,1H) 8.38 (d, J=8.8 Hz, 1H), 8.19-8.15 (m, 1H), 8.10-8.03 (m, 3H),7.93-7.88 (m, 1H), 7.76 (d, J=8.4 Hz, 1H), 7.69 (d, J=8.4 Hz, 2H),7.52-7.40 (m, 3H), 6.92 (t, J=7.6 Hz, 1H), 2.70-2.57 (m, 4H), 2.29-2.20(m, 1H), 1.90-1.80 (m, 1H); LCMS: 433 [M+H]. Calc. for C₂₉H₂₇ON₇ 3.06hydrochloric acid 0.01 dioxane 0.03 diethylether: C59.61, H5.28, N15.36.Found C59.62, H5.05, N15.36.

Synthesis of tert-butyl (1-(4-aminophenyl)cyclobutyl)carbamate, buildingblock 2 for example 21, General Procedure A:

Step 1 Cbz Protection of(1-(4-(((benzyloxy)carbonyl)amino)phenyl)cyclobutyl) carbamic acid

To a solution of 4-(1-((tert-butoxycarbonyl)amino)cyclobutyl)benzoicacid (15 g) in toluene (75 ml) and triethylamine (14.36 ml, 2 eq.) wasadded diphenyl phosphoryl azide (12.22 ml, 1.1 eq.). The reaction washeated to 100° C. and allowed to react for 2 hours, or until vigorousbubbling stopped. Benzyl alcohol (26.6 ml, 5 eq.) was added and thereaction was allowed to proceed at 100° C. for 2 hours. The reaction wascooled to room temperature and placed on an ice bath to cool. Followingthe precipitation of white solid from the reaction was allowed warm toroom temperature and stir overnight. Ether (200 ml) was added to thereaction and the product was filtered to give 13.1 g white solid. 400 MHz ¹H NMR (DMSO-d₆) δ: 9.55 (s, 1H), 7.44-7.24 (m, 10H), 5.14 (s, 2H),2.4-2.28 (m, 4H), 2.00-1.90 (m, 1H), 1.79-1.69 (m, 1H), 1.28 (bs, 9H);LCMS: 397 [M+H].

Step 2 tert-butyl (1-(4-aminophenyl)cyclobutyl)carbamate; intermediate 2for General Procedure A

A suspension of the cbz protected4-(1-((tert-butoxycarbonyl)amino)cyclobutyl) benzoic acid (9.545 g) inethyl acetate (125 mL) and methanol (125 mL) was heated until insolution. The solution was allowed to cool room temperature and 10% Pd/C(1.1 g) was added. The flask was charged with hydrogen and allowed toreact overnight at room temperature. Upon completion, the reaction wasfiltered through a pad of celite, and the celite washed with methanol(2×100 mL). The organics were concentrated under reduced pressure,giving the product as a colorless oil (6.3 g, 100%) which was used withno further purification. LCMS: 263 [M+H].

Example 2 Synthesis of3-(3-(4-(1-aminocyclobutyl)phenyl)-5-(3-morpholinophenyl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine

Step 1 tert-butyl(1-(4-((6-chloro-3-nitropyridin-2-yl)amino)phenyl)cyclobutyl) carbamate

To a solution of 2,6-dichloro-3-nitropyridine (5.11 g) in DMA (50 ml)and triethylamine (5 ml) chilled to 0° C. was added drop-wise a solutionof tert-butyl (1-(4-aminophenyl) cyclobutyl)carbamate (6.3 g) in DMA (25ml) over the course of 20 minutes. The reaction was allowed to stir at0° C. of one hour and then slowly allowed to warm to room temperatureand react overnight. Upon completion, the reaction was diluted withwater (250 mL) and extracted with ethyl acetate (2×200 ml). The organicswere combined, washed with saturated sodium bicarbonate solution (1×200ml), water (1×200 ml) and brine (1×100 ml). The organics were dried oversodium sulfate and concentrated under reduced pressure. Purification bycolumn chromatography (15% ethyl acetate in hexanes) gave the product asan orange solid (5.05 g, 50%). 400 M Hz ¹H-NMR (DMSO-d₆) δ: 10.05 (s,1H), 8.52 (d, J=8.8 Hz, 1H), 7.56-7.52 (m, 2H), 7.42-7.37 (m, 3H), 6.98(d, J=8.8 Hz, 1H), 2.47-2.34 (m, 4H), 2.04-1.96 (m, 1H), 1.84-1.74 (m,1H), 1.30 (bs, 9H); LCMS: 419 [M+H].

Step 2 tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate

To a solution of tert-butyl(1-(4-((6-chloro-3-nitropyridin-2-yl)amino)phenyl)cyclobutyl) carbamate(5.0 g) in anhydrous DMSO (60 ml) and anhydrous methanol (10 ml) wasadded 2-aminonicotinaldehyde (1.53 g) followed by Na₂S₂O₄ (6.25 g). Thereaction mixture was heated to 100° C. for 2 days. Upon completion ofthe reaction, water (250 ml) was added and the reaction was allowed tostir for 1 day at room temperature. The reaction was extracted withdichloromethane (2×200 ml). Upon extracting the second time, a largeamount of yellow solid precipitated from the water layer and the organiclayer. The solid was filtered off and found to be product. The productwas combined with the organic layers and dried under reduced pressure,giving the product as a yellow solid (3.1 g, 52%). 400 M Hz ¹H NMR(DMSO-d₆) δ: 8.26 (d, J=8.0 Hz, 1H), 8.00-7.96 (m, 1H), 7.69 (bs, 1H),7.54-7.35 (m, 5H), 7.24-7.08 (m, 1H), 7.04-6.96 (m, 2H), 6.32-6.28 (m,1H), 2.48-2.35 (m, 4H), 2.06-1.96 (m, 1H), 1.86-1.76 (m, 1H), 1.40-1.06(m, 9H); LCMS: 491 [M+H].

Step 3 tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-(3-morpholinophenyl)-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate

tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate(1 eq.) was suspended in a mixture of ethanol and toluene, 10 mL/mmolrespectively. A solution of NaHCO₃ sat. was added (3 mL/mmol). Thereaction mixture was degassed with nitrogen for 30 min. Pd(PPh₃)₄ (0.05eq.) and the boronic acid (1.1 eq.) were added to the reaction mixture.Subsequently it was heated to 100° C. overnight under nitrogen. Aftercooling down to room temperature it was diluted with dichloromethane (20mL/mmol) and water (10 mL/mmol). The organic phase was separated andwashed with brine (10 mL/mmol) and dried over Na₂SO₄. After filtrationthe solvent was removed in vacuo. The crude residue was purified bysilica gel chromatography (3-20% methanol in ethyl acetate).

Step 43-{3-[4-(1-aminocyclobutyl)phenyl]-5-(3-morpholin-4-ylphenyl)-3H-imidazo[4,5-b]pyridin-2-yl}pyridin-2-amine

The carbamate (1 eq.) was dissolved in methanol. HCl (20 eq., 4 M indioxane) was added and stirred at room temperature for 2 to 4 hours.Concentration of the solution under reduced pressure gave thede-protected amine (structure 7 as shown in Scheme 2) as hydrochloricacid salt, which was used for the next step without furtherpurification. 400 M Hz ¹H-NMR (DMSO-d₆) δ: 8.97-8.78 (m, 1H), 8.87 (brs, 2H), 8.67 (s, 1H), 8.45 (d, J=8.3 Hz, 1H), 8.41 (d, J=8.3 Hz, 1H),8.36 (s, 1H), 8.31-8.02 (m, 1H), 8.16 (dd, J=6.0 Hz and 1.8 Hz, 1H),7.87-7.80 (m, 1H), 7.77 (d, J=8.3 Hz, 2H), 7.69 (d, J=8.3 Hz, 2H),6.90-6.80 (m, 1H), 3.81-3.74 (m, 4H), 3.71-3.63 (m, 4H), 2.73-2.56 (m,4H), 2.31-2.17 (m, 1H), 1.94-1.79 (m, 1H); LCMS: 520 [M+H].

Example 3 Synthesis ofN-[1-(3-{3-[4-(1-aminocyclobutyl)phenyl]-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-5-yl}phenyl)piperidin-4-yl]-N-methylacetamidetrihydrochloride (Compound 3)

Step 1: synthesis ofN-methyl-N-{1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperidin-4-yl}acetamide

A mixture of N-[1-(3-bromophenyl)piperidin-4-yl]-N-methylacetamide (68mg, 0.217 mmol), bis(pinacolato)diboron (66 mg, 0.260 mmol),Pd(dppf)Cl₂·DCM (9 mg, 0.0109 mmol) and potassim acetate (64 mg, 0.651mmol) in dioxane (3 mL) was heated at 80° C. for 13 hours undernitrogen. After cooling to room temperature, the mixture was dilutedwith EtOAc and filtered through a Celite pad. The combined filtrate andwashings were concentrated. The residue was purified by silica gelcolumn chromatography (hexane/AcOEt=35:65→0:100) to affordN-methyl-N-(1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidin-4-yl)acetamide(61 mg, 78%) as white solid.

500 M Hz ¹H-NMR (CDCl₃) δ: 7.41-7.38 (m, 1H), 7.35-7.28 (m, 2H),7.06-7.03 (m, 1H), 4.68-4.61 (m, 1H), 3.84-3.76 (m, 2H), 2.88 (s, 2H),2.85 (s, 1H), 2.83-2.76 (m, 2H), 2.16 (s, 1H), 2.11 (s, 2H), 2.02-1.92(m, 1H), 1.83-1.76 (m, 2H), 1.72-1.69 (m, 1H), 1.34-1.34 (m, 12H); LCMS:359 [M+H].

Step 2 Coupling

A mixture of tert-butyl(1-{4-[2-(2-aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl]phenyl}cyclobutyl)carbamate(56 mg, 0.113 mmol),N-methyl-N-{1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperidin-4-yl}acetamide(61 mg, 0.170 mmol),bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II)(8 mg, 0.0113 mmol), and 2M Na₂CO₃ aq. (0.062 mL, 0.124 mmol) in DMF(2.5 mL) was treated with microwave (160° C. for 1 hour). The mixturewas diluted with AcOEt, then washed with water (×3), brine, dried overNa₂SO₄, then filtrated. The filtrate was concentrated and the residuewas purified by preparative thin-layer chromatography (AcOEt/MeOH=20:1),and further purified by preparative thin-layer chromatography(CH₂Cl₂/MeOH=20:1×2) to afford tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-(3-(4-(N-methylacetamido)piperidin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate(14 mg, 18%) as yellow solid.

Step 3 De-Protection

To tert-butyl(1-(4-(2-(2-aminopyridin-3-yl)-5-(3-(4-(N-methylacetamido)piperidin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)cyclobutyl)carbamate(14 mg, 0.0204 mmol) in MeOH (1 mL) was added 4N HCl-dioxane (3 mL) andstirred at r.t for 14 hours. The mixture was concentrated to affordN-[1-(3-{3-[4-(1-aminocyclobutyl)phenyl]-2-(2-aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-5-yl}phenyl)piperidin-4-yl]-N-methylacetamidetrihydrochloride (19 mg, quant) as pale yellow solid.

500 M Hz ¹H-NMR (DMSO-d₆) δ: 8.86-8.82 (m, 2H), 8.41-8.37 (m, 1H),8.36-8.23 (m, 2H), 8.27 (dd, J=10.0 Hz and 5.0 Hz, 1H), 8.15 (d, J=6.3Hz, 1H), 7.89-7.87 (m, 1H), 7.75 (dd, J=8.6 Hz and 2.9 Hz, 2H), 7.69 (d,J=8.6 Hz, 2H), 7.51-7.36 (m, 2H), 6.88 (t, J=6.9 Hz, 1H), 3.74-3.64 (m,2H), 3.50-3.45 (m, 3H), 2.85 (s, 2H), 2.70 (s, 1H), 2.68-2.57 (m, 4H),2.26-2.18 (m, 2H), 2.10 (s, 1H), 2.02 (s, 2H), 1.90-1.82 (m, 2H),1.82-1.80 (m, 1H), 1.67-1.60 (m, 2H); LCMS: 587 [M+H].

Example 4(R)-6-(2-fluorophenyl)-N-(3-(2-(2-methoxyethylamino)ethyl)phenyl)-5,6-dihydrobenzo[h]quinazolin-2-amine(Compound 4) hydrochloride salt

The compound was synthesized by using(R)-4-(6-(2-fluorophenyl)-5,6-dihydrobenzo[h]quinazolin-2-ylamino)phenethylmethanesulfonate, 2-methoxyethanamine and triethylamine as described ingeneral procedure 6 to afford the desired product. M.p.=173-175° C. ¹HNMR 400 MHz (DMSO-d₆) δ 9.68 (s, 1H), 8.99 (bs, 2H), 8.33-8.31 (m, 2H),7.73-7.69 (m, 2H), 7.54-7.44 (m, 2H), 7.29-7.24 (m, 3H), 7.06-7.00 (m,2H), 6.85-6.78 (m, 2H), 5.55 (bs, 2H), 4.65 (t, J=7.2 Hz, 1H), 3.61 (t,J=5.2 Hz, 2H), 3.29 (s, 3H), 3.20-3.08 (m, 6H), 2.98-2.94 (m, 2H). LCMSm/e 469 (M+H).

Example 5 Inactive AKT Alpha Screen Assay

AKT1 activity was assayed using the GSK3-derived biotinylated peptidesubstrate, crosstide (biotin-GRPRTSSFAEG), and AlphaScreen™ (AmplifiedLuminescent Proximity Homogeneous Assay) technology. AKT1 activation wasachieved by the addition of the activating kinases PDK1 and MAPKAPK2,lipid vesicles, and ATP. The extent of peptide phosphorylation wasdetermined using a phospho-AKT substrate antibody and acceptor beadsconjugated to Protein A and donor beads conjugated to streptavidin thatbind to the biotin on the peptide. Excitation of the donor beadsconverted ambient oxygen to excited singlet oxygen which, when in closeproximity to acceptor beads, reacted with acceptor beads resulting insignal amplification.

Test inhibitors and controls((S)-1-((5-(3-methyl-1H-indazol-5-yl)pyridin-3-yl)oxy)-3-phenylpropan-2-amine,1-(1-(4-(7-phenyl-1H-imidazo[4,5-g]quinoxalin-6-yl)benzyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one, and8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3(2H)-one) wereprepared in 10% DMSO at 10-fold the desired final concentration, andadded to each well of a reaction plate (Corning 96-well half-area solidwhite nonbinding surface plate) in a volume of 2.5 μL. Full-lengthinactive AKT1 was diluted in assay buffer (50 mM Tris, pH 8.0, 0.02mg/mL BSA, 10 mM MgCl₂, 1 mM EGTA, 10% glycerol, 0.2 mM Na₃VO₄, 1 mMDTT, 0.1 mM β-glycerophosphate, and 0.2 mM NaF) and added to each wellin a volume of 17.5 μL for a final concentration in the 25 μL reactionof 8 nM (AKT1). After a 20 minute pre-incubation at room temperature,the kinase reaction was initiated by the addition of 5 μL of anactivation mixture diluted in assay buffer containing biotinylatedcrosstide, PDK1, MAPKAPK2, DOPS/DOPC, PtdIns(3,4,5)P3, and ATP for finalconcentrations of 60 nM biotinylated crosstide, 0.1 nM PDK1, 0.7 nM MK2,5.5 μM DOPS, 5.5 μM DOPC, 0.5 μM PtdIns(3,4,5)P3, and 50 μM ATP. Theplates were incubated for 30 minutes at room temperature, and thenstopped in the dark by the addition of 10 μL stop/detection mixtureprepared in assay buffer containing EDTA, AlphaScreen™ StreptavidinDonor and Protein A Acceptor beads, and phospho-AKT substrate antibodyfor final concentrations of 10 mM EDTA, 500 ng/well of both AlphaScreen™Streptavidin Donor beads and Protein A Acceptor beads, and phospho-AKTsubstrate antibody at a final dilution of 1:350. Assay plates wereincubated for 90 minutes at room temperature in the dark, and the plateswere read on a Perkin Elmer Envision Multilabel plate reader (excitationwavelength: 640 nm, emission wavelength: 570 nm).

Reaction:

-   -   2.5 μL 10×AKT inhibitor in 10% DMSO    -   17.5 μL inactive AKT or buffer for blank    -   20 minute pre-incubation at room temperature    -   5 μL Reaction Mix (5×ATP, 5× substrate, 5×PDK1, 5× MK2, and 5×        lipid vesicle mixture)    -   30 minute incubation at room temperature    -   10 μL Detection Buffer    -   90 minute incubation at room temperature    -   Detection (excitation: 640 nm, emission: 570 nm)

Envision Instrument Settings:

-   -   Instrument: Perkin Elmer Envision    -   Plate: 96 well    -   Program Name:    -   Excitation: Ex Top    -   Mirror: General Dual-Slot 2    -   Excitation Filter: CFP430 Ex. Slot 2    -   Emission Filter: Emission 579-Em slot 2    -   2nd Emission Filter: None    -   Measurement Height (mm): 3.8    -   Excitation light (%): 1    -   Detector gain: 1    -   2nd detector gain: 0    -   # Flashes: 10    -   # Flashed/AD: 1    -   Reference Signal: 383722    -   AD gain: 4    -   Reference Excitation (%): 100

Example 6 Inactive AKT HTRF Assay

AKT1 activity was assayed using CisBio KinEASE™ HTRF Assay technology.This technology utilizes a proprietary biotinylated peptide substrate(STKS3), streptavidin labeled XL665 antibody, and STKantibody-Eu³⁺-cryptate. AKT1 activation was achieved by the addition ofthe activating kinases PDK1 and MAPKAPK2, lipid vesicles, and ATP. Theextent of STKS3 biotinylated peptide phosphorylation was determinedusing a phospho-STK antibody-Eu³⁺-cryptate and streptavidin labeledXL665 antibody. XL665 was stimulated by Eu³⁺-cryptate resulting in aTR-FRET signal proportional to the SKS3 phosphorylation level.

Test inhibitors and controls((S)-1-((5-(3-methyl-1H-indazol-5-yl)pyridin-3-yl)oxy)-3-phenylpropan-2-amine,1-(1-(4-(7-phenyl-1H-imidazo[4,5-g]quinoxalin-6-yl)benzyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one, and8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3(2H)-one) wereprepared in 10% DMSO at 10-fold the desired final concentration, andadded to each well of a reaction plate (Corning 96-well half-area solidblack nonbinding surface plate) in a volume of 2.5 μl. Full-lengthinactive AKT1, AKT2, and AKT3 were diluted in assay buffer (50 mM Tris,pH 8.0, 0.02 mg/ml BSA, 10 mM MgCl₂, 1 mM EGTA, 10% glycerol, 0.2 mMNa₃VO₄, and 1 mM DTT) and added to each well in a volume of 17.5 μl fora final concentration in the 25 μl reaction of 8 nM (AKT1), 20 nM(AKT2), or 3 nM (AKT3). After a 20 minute pre-incubation at roomtemperature, the kinase reaction was initiated by the addition of 5 μlofan activation mixture diluted in assay buffer containing biotinylatedSTKS3, PDK1, MAPKAPK2, DOPS/DOPC, PtdIns(3,4,5)P3, and ATP for finalconcentrations of 150 nM biotinylated STKS3, 1 nM (AKT1), 2.5 nM (AKT2),or 0.4 nM (AKT3) PDK1, 0.8 nM (AKT1), 2 nM (AKT2), or 0.3 nM (AKT3) MK2,5.5 μM DOPS, 5.5 μM DOPC, 0.5 μM PtdIns(3,4,5)P3, and 50 μM ATP. Theplates were incubated for 30 minutes at room temperature, and thenstopped by the addition of 25 μl HTRF Detection Buffer containingphospho-STK antibody-Eu³⁺-cryptate and streptavidin labeled XL665antibody, at dilutions of 1:192 and 1:500 respectively. Final assaydilutions of phospho-STK antibody-Eu³⁺-cryptate and streptavidin labeledXL665 antibody were 1:384 and 1:1,000 respectively. Assay plates wereincubated for 60 minutes at room temperature, and the plates were readon a Perkin Elmer Envision Multilabel plate reader (excitation: 320 nm,emission I: 665 nm, emission II: 615 nm).

Reaction:

-   -   2.5 μl 10×AKT inhibitor in 10% DMSO    -   17.5 μl inactive AKT or buffer for blank    -   20 minute pre-incubation at room temperature    -   5 μl Reaction Mix (5×ATP, 5× substrate, 5×PDK1, 5× MK2, and 5×        lipid vesicle mixture)    -   30 minute incubation at room temperature    -   25 μl Detection Buffer    -   60 minute incubation at room temperature    -   Detection (excitation: 320 nm, emission I: 665 nm, emission II:        615 nm)

Example 7 MTS Assay

Cell proliferation analysis. Cell survival was determined by the MTSassay. Briefly, cells were plated in a 96-well plate at 2,000-15,000cells per well, cultured for 24 hours in complete growth medium, andthen treated with various drugs and drug combinations for 72 hours. MTSand PMS reagent were added and incubated for 4 hours, followed byassessment of cell viability using the microplate reader at 490 nm. Datawere normalized to untreated controls and analyzed with Microsoft Excel.

Table 1 shows the physical property of Compound 1, Compound 2 andCompound 3.

TABLE 1 Com- pound Chemical Name [M + H] 13-(3-(4-(1-aminocyclobutyl)phenyl)-5-phenyl- 4333H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine 23-(3-(4-(1-aminocyclobutyl)phenyl)-5-(3-morpho- 518linophenyl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin- 2-amine 3N-(1-(3-(3-(4-(1-aminocyclobutyl)phenyl)-2-(2- 587aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-5-yl)phenyl)piperidin-4-yl)-N-methylacetamide

Table 2 shows the AKT kinase inhibition activity of Compound 1, Compound2 and Compound 3.

TABLE 2 AKT1 IC₅₀, (Alpha- Com- Screen) pound Chemical Name (μM) 13-(3-(4-(1-aminocyclobutyl)phenyl)-5-phenyl- 0.00323H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine 23-(3-(4-(1-aminocyclobutyl)phenyl)-5-(3-morpho- 0.0022 linophenyl)-3H-imidazo[4,5-b]pyridin-2-yl)pyridin- 2-amine 3N-(1-(3-(3-(4-(1-aminocyclobutyl)phenyl)-2-(2- 0.0020aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-5-yl)phenyl)piperidin-4-yl)-N-methylacetamide

Table 3 shows the MTS activity of Compound 1.

TABLE 3 Average of [CELL], IC50, MTS (72 hours; 10 Percent; FBS) (μM)A2780 AN3CA BT474 LnCap IGROV Zr75-1 0.72 0.62 1.3 2.1 1.7 4.2

For combination studies, cells were seeded in 96-well tissue-cultureplates at optimal number cells per well overnight and subsequentlytreated with serial dilutions of Compound 1 with serial dilutions ofCompound 4. The starting concentration for both agents was determinedbased on GI₅₀ for the single agent. Treated cells were incubated at 37°C. for 72 hrs in 5% CO₂.

Thirty microliters of the mixture of MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfphenyl)-2Htetrazolium))reagent (18.4 mg/ml) and PMS (Phenazine methosulfate) (0.92 mg/ml) at aratio of 20:1 was added to each well, and the plates were incubated at37° C. for 4 hrs in 5% CO₂. The absorbance was measured at 490 nM usingthe micromicroplate reader.

For combination study, The Combination Index (CI) was determined usingthe Chou-Talalay method. Synergistic: CI≦0.85; Additive: CI≧0.85 and≦1.2; and Antagonistic: CI≧1.2.

Forty-five cell lines representing thirteen cancer types were tested forcombination of Compound 1 and Compound 4: ovary, endometrial, CRC,Bladder, triple negative breast cancer, CNS, lymphoma/leukemia, lung,and prostate. Compound 1 and Compound 4 are combinable showing 24%(11/45) synergistic; 62% (28/45) additive; and only 13% (6/45)antagonistic. Ovarian and endometrial cancers have higher synergism rateof 50% (3/6) and 67% (4/6) respectively. Furthermore, there is 33% (2/6)synergism in triple negative breast cancer cell lines.

The results are shown in Table 4.

TABLE 4 Combination GI₅₀ (mM) Cells Type CI (N) Effect Compound 1Compound 4 IGROV-1 Ovary 0.59 ± 0.21(5) Synergistic  0.27 ± 0.26(10) 1.26 ± 0.57(10) SKOV-3 Ovary 0.80 ± 0.12(6) Synergistic  4.22 ±1.50(11)  4.41 ± 0.76(11) CaoV-3 Ovary 0.98 ± 0.08(3) Additive 6.66 ±1.57(7) 3.12 ± 0.68(7) OVCAR3 Ovary 0.82 ± 0.08(5) Synergistic 0.40 ±0.19(5) 4.17 ± 0.68(5) TOV21G Ovary 0.92 ± 0.11(3) Additive 8.62 ±4.07(2) 3.69 ± 1.17(2) MCAS Ovary 0.92 ± 0.05(2) Additive 6.37 ± 2.45(4)6.96 ± 3.00(4) OVMANA Ovary 1.01 ± 0.06(2) Additive 4.21 ± 0.93(2) 4.26± 0.11(2) OVISE Ovary 1.34 ± 0.12(3) Antagonistic 2.20 ± 0.81(3) 6.24 ±1.45(3) AN3CA Endometrial 0.48 ± 0.20(4) Synergistic  0.75 ± 0.24(10)1.03 ± 0.25(8) MFE296 Endometrial 0.43 ± 0.16(4) Synergistic 0.82 ±0.38(8) 3.22 ± 0.42(8) RL95-2 Endometrial 1.00 ± 0.00(2) Additive 2.06 ±0.47(3) 2.53 ± 0.64(3) MFE-280 Endometrial 0.70 ± 0.10(4) Synergistic0.11 ± 0.08(7) 1.17 ± 0.32(7) HEC-1B Endometrial 0.95 ± 0.15(2) Additive4.09 ± 0.77(4) 5.27 ± 2.74(4) EN Endometrial 0.67 ± 0.13(3) Synergistic1.33 ± 0.57(4) 2.08 ± 0.78(6) Ishikawa Endometrial 0.91 ± 0.06(3)Additive 5.40 ± 0.36(2) 3.49 ± 0.40(2) HEC-251 Endometrial 0.96 ±0.06(3) Additive 15.2 ± 0.24(3) 10.3 ± 0.98(3) SNG-II Endometrial 0.90 ±0.12(4) Additive 5.85 ± 0.83(3) 3.71 ± 0.40(3) C-33A Cervix 1.24 ±0.18(3) Antagonistic 4.78 ± 0.62(2) 4.28 ± 0.19(2) Ca Ski Cervix 1.27 ±0.05(3) Antagonistic 5.82 ± 0.68(2) 6.38 ± 1.80(2) ME-180 Cervix 1.07 ±0.13(3) Additive 1.00 ± 0.48(3) 4.10 ± 0.25(3) SW962 Vulvar 1.03 +0.04(2) Additive 4.41 ± 0.17(3) 3.15 ± 0.52(3) HCT116 CRC 0.90 ± 0.17(4)Additive 7.56 ± 1.64(6) 4.82 ± 0.08(6) DLD1 CRC 0.97 ± 0.13(4) Additive13.4 ± 1.31(6) 4.50 ± 0.36(6) HT-1197 Bladder 1.01 ± 0.16(2) Additive1.04 ± 0.34(3) 1.78 ± 0.23(3) 639-V Bladder 0.92 ± 0.07(2) Additive 4.58± 0.05(3) 2.30 ± 0.34(3) TCCSUP Bladder 0.78 ± 0.08(5) Synergistic 1.62± 0.31(3) 3.36 ± 0.92(4) MDA-MB-468 Breast 1.08 ± 0.09(3) Additive 1.34± 0.41(3) 3.76 ± 0.12(3) MDA-MB-231 Breast 0.93 ± 0.11(3) Additive8.33 + 1.94(4) 4.05 + 0.31(4) BT549 Breast 0.89 ± 0.00(2) Additive 3.10± 0.56(3) 3.26 ± 0.54(3) BT20 Breast 0.83 ± 0.10(4) Synergistic 5.75 ±2.77(7) 3.92 ± 0.27(7) Cal-51 Breast 0.60 ± 0.09(4) Synergistic 3.85 ±0.63(3) 1.40 ± 0.62(4) MDA-MB-453 Breast 1.05 ± 0.11(4) Additive 1.23 ±0.65(4) 2.71 ± 0.98(4) REC-1 Lymphoma 1.06 ± 0.06(2) Additive 9.03 ±1.15(3) 3.97 ± 0.34(3) Jurkat Leukemia 1.17 ± 0.14(4) Additive 0.21 ±0.14(3) 1.35 ± 011(3)  NCI-H460 Lung 0.96 ± 0.06(4) Additive 12.53 ±8.84(4)  3.21 ± 1.78(4) NCI-H596 Lung 0.64 ± 0.14(3) Synergistic 6.87 ±0.22(2) 9.02 ± 1.03(2) 22Rv1 Prostate 1.44 ± 0.11(4) Antagonistic 0.98 ±0.36(7) 5.08 ± 1.55(3) A-172 CNS 1.03 ± 0.1(3)  Additive 6.79 ± 1.48(7)2.68 ± 0.43(7) U87MG CNS 0.88 ± 0.22(4) Additive 11.35 ± 2.49(4)  3.90 ±0.47(4) IMR-32 CNS 1.21 + 0.24(2) Additive 4.79 ± 0.42(2) 2.74 + 1.08(2)SK-N-AS CNS 0.86 + 0.07(3) Additive 11.3 + 2.30(4) 5.78 + 0.99(4)SK-N-SH CNS 1.11 + 0.09(2) Additive 4.52 ± 0.03(1) 2.81 ± 0.41(1) SCC-25Head and Neck 1.25(1) Antagonistic 1.34 ± 0.85(2) 3.38 ± 0.39(2) SCC-9Head and Neck 1.08 + 0.10(2) Additive 5.37 ± 0.80(2) 3.82 ± 0.69(2) FaduHead and Neck 1.05 + 0.09(3) Additive 8.95 ± 1.75(4) 4.15 ± 0.17(4)

Example 8 Proteus Syndrome

The compounds of the present invention, alone or in combination, can beutilized in the treatment of Proteus syndrome.

FIG. 1 shows the viability of Proteus cells in the presence of serum andvarious dosages of Compound 1. Cells were plated and permitted to attachovernight. Cells were refed with normal medium containing Compound 1 andthen harvested 72 hours later. Mutation positive and mutational negativecell lines from Proteus Syndrome patients (Referred to in FIG. 1 as 75.2pos 1, pos 2, neg 1, neg 2; 53.3 pos 1, pos 2, neg 1, neg 2). The 1 and2 refer to experiments (the same cell line was tested twice). F6B posand H4A neg are single cell clones from patient 134.3 cell line.Viability was measured using the CellTiterGlo cell viability assay fromPromega. Each data point is an average of 3 wells (technical replicate)and each line is a biological replicate. The mutation negative cells allhave higher viability until 2.5 uM.

FIG. 2 shows the viability of Proteus cells in the presence of low serumand various dosages of Compound 1. Cells were plated and permitted toattach overnight. Cells were washed and refed with 0.5% serum medium for24 hours. Cells were then washed and refed with 0.5% medium containingCompound 1 and harvested 72 hours later.

FIG. 3 shows the viability of PIK3CA cells in the presence of serum andvarious dosages of Compound 1. Cells were plated and permitted to attachovernight. Cells were refed with normal medium containing Compound 1 andthen harvested 72 hours later. Mutation positive cell lines (Referred toin FIG. 3 as 109.3 pos and 110.3 pos) from PIK3CA patients andmutational negative cell lines (Referred to in FIG. 3 as 95.1 neg and95.2 neg) from non-OG control individuals. The mutation positive cellsare more sensitive down to 1.25 uM.

FIG. 4 shows the viability of PIK3CA cells in the presence of low serumand various dosages of Compound 1. Cells were plated and permitted toattach overnight. Cells were washed and refed with 0.5% serum medium for24 hours. Cells were then washed and refed with 0.5% medium containingCompound 1 and harvested 72 hours later. Results indicate that mutationpositive cells are more sensitive than control cells.

FIGS. 5A and 5B show the viability of Proteus single cell clones (A6BAKT1 p.E17K positive and E8F9A mutation negative cells) in the presenceor absence of serum and various dosages of Compound 1 (FIG. 5A) oreverolimus (FIG. 5B). Cells were plated and permitted to attachovernight. Cells were washed and refed with 0.5% serum medium for 24hours. Cells were then washed and refed with 0.5% medium or normalmedium containing Compound 1 or everolimus and harvested 72 hours later.Results indicate that mutation positive cells are more sensitive thancontrol cells.

FIG. 6 shows the phosphorylation status of AKT1 in Proteus single cellclones (A6B AKT1 p.E17K positive and E8F9A mutation negative cells) inthe presence or absence of serum and various dosages of Compound 1.Cells were plated and permitted to attach overnight. Cells were washedand refed with 0.5% serum medium for 24 hours. Cells were then washedand refed with 0.5% medium or normal medium containing Compound 1 for 24hours. Cell lysates were then analyzed for AKT1 phosphorylation status.The results show a decrease in phosphorylation with increasing dose;however wild type cells have minimal pAKT signal in the absence of seruminitially.

FIGS. 7A and 7B show the phosphorylation status of S6 in Proteus singlecell clones (A6B AKT1 p.E17K positive and E8F9A mutation negative cells)in the presence (FIG. 7B) or absence of serum (FIG. 7A) and variousdosages of Compound 1. Cells were plated and permitted to attachovernight. Cells were washed and refed with 0.5% serum medium for 24hours. Cells were then washed and refed with 0.5% medium or normalmedium containing Compound 1 for 24 hours. Cell lysates were thenanalyzed for S6 phosphorylation status. The results show that Compound 1does not appear to have an effect on pS6 levels in cells grown in serumfree medium and has only a slight affect in cells grown in normalmedium.

FIGS. 8A and 8B show the phosphorylation status of AKT1 in fourdifferent Proteus cell lines from a single patient with differing AKT1p.E17K in the presence (FIG. 8A) or absence of serum (FIG. 8B) andvarious dosages of Compound 1. Cells were plated and permitted to attachovernight. Cells were washed and refed with 0.5% serum medium for 24hours. Cells were then washed and refed with 0.5% medium or normalmedium containing Compound 1 for 24 hours. Cell lysates were thenanalyzed for AKT1 phosphorylation status. The results show a decrease inphosphorylation with increasing dose, which is especially apparent incell line with the high levels of AKT1 p.E17K.

FIGS. 9A and 9B show the phosphorylation status of S6 in four differentProteus cell lines from a single patient with differing AKT1 p.E17K inthe presence (FIG. 9B) or absence of serum (FIG. 9A) and various dosagesof Compound 1. Cells were plated and permitted to attach overnight.Cells were washed and refed with 0.5% serum medium for 24 hours. Cellswere then washed and refed with 0.5% medium or normal medium containingCompound 1 for 24 hours. Cell lysates were then analyzed for S6phosphorylation status. The results do not indicate any specific effectof Compound 1 on pS6 in these cell lines.

FIG. 10 shows the phosphorylation status of AKT1 of cells obtained froma patient with PIK3CA p.H1047R mutation (PS109.3) or control cells(PS95.2) in the presence or absence of serum and various dosages ofCompound 1. Cells were plated and permitted to attach overnight. Cellswere washed and refed with 0.5% serum medium for 24 hours. Cells werethen washed and refed with 0.5% medium or normal medium containingCompound 1 for 24 hours. Cell lysates were then analyzed for AKTphosphorylation status. The results show a decrease in phosphorylationwith increasing dose.

FIGS. 11A and 11B show the phosphorylation status of S6 of cellsobtained from a patient with PIK3CA p.H1047R mutation (PS109.3) orcontrol cells (PS95.2) in the presence (FIG. 11B) or absence of serum(FIG. 11A) and various dosages of Compound 1. Cells were plated andpermitted to attach overnight. Cells were washed and refed with 0.5%serum medium for 24 hours. Cells were then washed and refed with 0.5%medium or normal medium containing Compound 1 for 24 hours. Cell lysateswere then analyzed for S6 phosphorylation status. The results show thatCompound 1 has a modest affect on mutation positive cells both with andwithout serum.

FIG. 12 shows the phosphorylation status of AKT1 of cells obtained froma patient with PIK3CA p.H1047L mutation (PS129.3, GSA) or control cells(PS75.1) in the presence or absence of serum and various dosages ofCompound 1. Cells were plated and permitted to attach overnight. Cellswere washed and refed with 0.5% serum medium for 24 hours. Cells werethen washed and refed with 0.5% medium or normal medium containingCompound 1 for 24 hours. Cell lysates were then analyzed for AKTphosphorylation status. The results show the same profile as p.H105Rmutant cells.

FIGS. 13A and 13B show the phosphorylation status of S6 of cellsobtained from a patient with PIK3CA p.H1047L mutation (PS129.3, GSA) orcontrol cells (PS75.1) in the presence (FIG. 13B) or absence of serum(FIG. 13A) and various dosages of Compound 1. Cells were plated andpermitted to attach overnight. Cells were washed and refed with 0.5%serum medium for 24 hours. Cells were then washed and refed with 0.5%medium or normal medium containing Compound 1 for 24 hours. Cell lysateswere then analyzed for S6 phosphorylation status. The results show thatCompound 1 has a modest affect on mutation positive cells both with andwithout serum.

FIGS. 14A, 14B, 14C, and 14D show the phosphorylation status of AKT1 inProteus single cell clones (F6B AKT1 p.E17K positive and H4A mutationnegative cells) in the presence (FIGS. 14C and 14D) or absence of serum(FIGS. 14A and 14B) and 125 nM of Compound 1. Cells were plated andpermitted to attach overnight. Cells were washed and refed with 0.5%serum medium for 24 hours. Cells were then washed and refed with 0.5%serum medium or normal medium containing 125 nM of Compound 1. Cellswere harvested at the times indicated and lysates were analyzed for AKT1phosphorylation status.

FIG. 15 shows the phosphorylation status of AKT1 in Proteus single cellclones (F6B AKT1 p.E17K positive and H4A mutation negative cells) in thepresence or absence of serum and various dosages of everolimus. Cellswere plated and permitted to attach overnight. Cells were washed andrefed with 0.5% serum medium for 24 hours. Cells were then washed andrefed with 0.5% serum medium or normal medium containing the variousdosages of everolimus. Cell lysates were analyzed for AKT1phosphorylation status. The results show that everolimus decreases thepAKT/AKT ratio in mutant cells in serum free conditions, but has noeffect or increases the ratio in mutant negative cells or in mutantcells gown in serum free conditions.

FIGS. 16A and 16B show the phosphorylation status of S6 in Proteussingle cell clones (F6B AKT1 p.E17K positive and H4A mutation negativecells) in the presence (FIG. 16B) or absence of serum (FIG. 16A) andvarious dosages of everolimus. Cells were plated and permitted to attachovernight. Cells were washed and refed with 0.5% serum medium for 24hours. Cells were then washed and refed with 0.5% serum medium or normalmedium containing the various dosages of everolimus. Cell lysates wereanalyzed for S6 phosphorylation status. The results show that everolimusgreatly decreases pS6 levels in both mutation positive and mutationnegative Proteus single cell clones.

FIG. 17 shows the phosphorylation of AKT in KU-19-19 (E17K) mutantbladder cancer cells and AN3CA endometrial cancer cells followingtreatment with various dosages of Compound 1. Specifically, cells fedwith normal medium comprising various dosages of Compound 1 for 2 hours.Proteins were detected with the following antibodies:pAKT(T473)(CST#4060); pAKT(S308)(CST#2965); AKT1(CST#2967);AKT(pan)(CST#2920); pPRAS40(T246)(CST#2997). The results show thatCompound 1 inhibits pAKT and pPRAS40 (phosphorylated proline-rich AKTsubstrate of 40 kDa) in KU-19-19 and AN3CA cells.

FIG. 18 shows the phosphorylation of AKT in KU-19-19 (E17K) mutantbladder cancer cells following treatment with various dosages ofCompound 1, MK-2206 (an allosteric AKT inhibitor) and GDC0068 (a selectATP-competitive pan-AKT inhibitor). Specifically, cells fed with normalmedium comprising various dosages of Compound 1 for 2 hours. Proteinswere detected with the following antibodies: pAKT(T473)(CST#4060);AKT(pan)(CST#2920); pPRAS40(T246)(CST#2997); pERK (T202/Y204)(CST#4370).The results show that Compound 1 and MK-2206, but not GDC0068, inhibitpAKT and pPRAS40 in KU-19-19 cells.

Example 9 Dose-Escalation Study

Eighty-two subjects with advanced solid tumors or recurrent malignantlymphoma were treated with Compound 1 in a dose-escalation study.Preliminary signals of single agent activity with advanced tumors wereobserved, including one partial response in a heavily pretreatedlymphoma subject. The overall disease control rate, including partialresponses, minor responses and stable disease, was 34.1%. Reductions inthe the expression levels of relevant biomarkers were noted followingtreatment.

A manageable safety profile in cancer patients was defined consistentwith pre-clinical models and with other AKT inhibitors. Drug exposure inpatients was shown to increase in a dose-dependent fashion. Maximumtolerated doses (MTDs) and recommended Phase 2 doses were establishedfor continuous (60 milligrams daily), intermittent (200 milligrams dailyevery other week) and weekly dosing (300 milligrams once a week)schedules.

We claim:
 1. A method of treating a cell proliferative disorder, saidmethod comprising administering, to a subject in need thereof, atherapeutically effective amount of a composition comprising at leastone of

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof, wherein said cell proliferative disorder is treated.
 2. Themethod of claim 1, wherein said cell proliferative disorder is aprecancerous condition.
 3. The method of claim 1, wherein said cellproliferative disorder is a hematologic tumor or malignancy.
 4. Themethod of claim 1, wherein said cell proliferative disorder is a solidtumor.
 5. The method of claim 1, wherein said cell proliferativedisorder is a cancer.
 6. The method of claim 5, wherein said cancer islung cancer, small cell lung cancer, non-small cell lung cancer, coloncancer, breast cancer, pancreatic cancer, prostate cancer, anal cancer,renal cancer, cervical cancer, brain, gastric/stomach cancer, head andneck cancer, thyroid cancer, bladder cancer, endometrial cancer, uterinecancer, intestinal cancer, hepatic cancer, leukemia, lymphoma, T-celllymphoblastic leukemia, primary effusion lymphoma, chronic myelogenousleukemia, melanoma, Merkel cell cancer, ovarian cancer, alveolar softpart sarcoma (ASPS), clear cell sarcoma (CCS), Paget's disease,rhabdomysarcoma, angiosarcoma, cholangiocarcinoma or hepatocellularcarcinoma.
 7. The method of claim 5, wherein said cancer is a metastaticcancer.
 8. The method of claim 1, wherein said cell proliferativedisorder is a non-cancer disorder.
 9. The method of claim 8, whereinsaid non-cancer disorder is pituitary adenoma, leishmaniasis,skin-related hyperproliferative disorders, psoriasis, eczema,hyperpigmentation disorders, eye-related hyperproliferative disorders,age-related macular degeneration, Herpes simplex virus, Proteus syndrome(Wiedemann syndrome), macrodactyly syndrome, Harlequin ichthyosis,CLOVES syndrome, atopic dermatitis, LEOPARD syndrome, systemicsclerosis, Spinocerebullar ataxia type 1, fibroadipose hyperplasia,hemihyperplasia-multiple lipomatosis syndrome, megalencephaly, rarehypoglycemia, Klippel-Trenaunay syndrome, harmatoma, Cowden syndrome orovergrowth-hyperglycemia.
 10. The method of claim 9, wherein saidnon-cancer disorder is Proteus syndrome.
 11. The method of claim 1,wherein said subject is a human.
 12. The method of claim 5, wherein saidtreating cancer comprises a reduction in tumor size, inhibition ofmetastatic cancer cell invasion or both.
 13. The method of claim 1,wherein the composition is administered intravenously, orally orintraperitoneally.
 14. The method of claim 1, wherein the compositionsfurther comprise one or more pharmaceutically acceptable carriers orexcipients.
 15. The method of claim 1, wherein the composition isadministered daily.
 16. The method of claim 15, wherein the compositionis administered at about 50 mg to about 100 mg daily.
 17. The method ofclaim 16, wherein the composition is administered at about 60 mg daily.18. The method of claim 1, wherein the composition is administered in anintermittent dosing regimen, wherein the composition is administered atleast once in 24 hours, not administered for at least six days, andadministered at least once in 24 hours following the at least six days.19. The method of claim 18, wherein the composition is administered oncea week.
 20. The method of claim 18, wherein the composition isadministered once at about 250 mg to about 350 mg.
 21. The method ofclaim 20, wherein the composition is administered at about 200 mg daily.22. The method of claim 20, wherein the composition is administered onceat about 300 mg.
 23. The method of claim 1, wherein the composition isadministered in an intermittent dosing regimen, wherein the compositionis administered at least once daily for at least a week, notadministered for at least a second week, and administered daily for atleast a third week following the second week.
 24. The method of claim23, wherein the composition is administered at about 150 mg to about 250mg daily.
 25. The method of claim 1, further comprising administering atherapeutically effective amount of an additional anti-proliferativeagent, administering radiation therapy or both.
 26. The method of claim25, wherein the additional anti-proliferative agent is a kinaseinhibitor, an alkylating agent, an antibiotic, an anti-metabolite, adetoxifying agent, an interferon, a polyclonal or monoclonal antibody, aHER2 inhibitor, a histone deacetylase inhibitor, a hormone, a mitoticinhibitor, an MTOR inhibitor, a taxane or taxane derivative, anaromatase inhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, or a cytidine analogue drug.
 27. The methodof claim 25, wherein the additional anti-proliferative agent is afibroblast growth factor receptor inhibitor.
 28. The method of claim 25,wherein the additional anti-proliferative agent is a compositioncomprising

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof.
 29. The method of claim 28, wherein the composition comprising

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof is administered simultaneously with, preceding administrationof, or following administration of the composition comprising

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof.
 30. The method of claim 28, wherein the composition comprising

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug isadministered within 24 hours of administration of the compositioncomprising

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof.
 31. A pharmaceutical composition comprising a therapeuticallyeffective amount of a

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof, and a therapeutically effective amount of

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof, and one or more pharmaceutically acceptable carriers orexcipients.
 32. A kit for the treatment of a cell proliferative disorderin a subject comprising at least two separate vials, a first vialcomprising a therapeutically effective amount of a compositioncomprising

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof, and a second vial comprising a therapeutically effective amountof a composition comprising

or a pharmaceutically acceptable salt, solvate, hydrate, or prodrugthereof, with instructions for administering said composition in saidfirst vial and said composition in said second vial.